Engineered biocatalysts useful for carbapenem synthesis

ABSTRACT

The present disclosure provides engineered pNB esterase polypeptides useful for the synthesis of the carbapenem antibiotic, imipenem. The disclosure also provides polynucleotides encoding the engineered pNB esterases, host cells capable of expressing the engineered pNB esterases, and methods of using the engineered pNB esterases in the production of imipenem.

The present application is a Divisional of co-pending U.S. patentapplication Ser. No. 14/760,963, field on Jul. 14, 2015, which is anational stage application filed under 35 USC §371 and claims priorityto international application to PCT International Application No.PCT/US2014/011767, filed Jan. 16, 2014 which claims priority to U.S.Provisional Appln. Ser. No. 61/754,095, filed Jan. 18, 2013, each ofwhich is incorporated for all purposes in its entirety.

1. TECHNICAL FIELD

The disclosure relates to pNB esterase biocatalysts and processes usingthe biocatalysts for the preparation of the carbapenem antibioticcompound, imipenem.

2. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of “CX2-128USP1_ST25.txt”, a creation date of Jan. 17, 2013,and a size of 369,801 bytes. The Sequence Listing filed via EFS-Web ispart of the specification and is incorporated in its entirety byreference herein.

3. BACKGROUND

Carbapenems are a class of β-lactam antibiotics with a broad spectrum ofantibacterial activity. Carbapenem antibiotics were originally developedfrom thienamycin, a naturally derived product of Streptomyces cattleya.They have a structure that is highly resistant to most β-lactamases andconsequently, are often the antibiotic of last resort for treatment ofhighly resistant infections of bacteria such as Escherichia coli andKlebsiella pneumonia. Carbapenem antibiotics include, but are notlimited to, imipenem, ertapenem, meropenem, doripenem, panipenem,biapenem, razupenem, and tabipenem.

Imipenem, (compound (1) shown below), is a carbapenem that exhibits abroad range of antibiotic activity against gram-positive andgram-negative aerobic and anaerobic bacteria species.

The methods of use and manufacture of imipenem were first disclosed inU.S. Pat. No. 4,194,047. Various alternative synthetic methods for thepreparation of imipenem are described in e.g., U.S. Pat. Nos. 4,374,772,4,894,450, and 7,462,712. Several synthesis routes for the preparationof imipenem include the formation of a p-nitrobenzyl-ester of imipenem(compound (2) shown below).

This pNB-protected imipenem of compound (2) is deprotected to providethe imipenem product of compound (1) by using palladium or platinumhydrogenation catalysts to remove the remove p-nitrobenzyl group (seee.g., U.S. Pat. Nos. 4,292,436, and 5,245,069, and U.S. Pat. Publ.2002/0095034). The use of these catalysts for pNB deprotection, however,is expensive and results in relatively low yields of the deprotectedimipenem (e.g., <60%).

The use of an esterase enzyme to remove pNB protecting groups in thesynthesis of the cephalosporin-derived and 1-carba-cephalasporinantibiotics was disclosed in U.S. Pat. No. 5,468,632. A specificwild-type pNB esterase from Bacillus subtilis for removing such pNBprotecting groups was isolated, cloned, and sequenced in U.S. Pat. No.5,468,632. This same wild-type pNB esterase also has been engineered forincreased thermostability and activity in the removal of the pNB groupfrom a pNB-protected precursor of the antibiotic Loracarbef (see e.g.,Moore et al., “Directed evolution of a para-nitrobenzyl esterase foraqueous-organic solvents,” Nature Biotechnology 14: 458-467 (1996);Moore et al. “Strategies for the in vitro Evolution of Protein Function:Enzyme Evolution by Random Recombination of Improved Sequences,” J. Mol.Biol. 272:336-347 (1997); Giver et al., “Directed evolution of athermostable esterase,” Proc. Natl. Acad. Sci. USA 95: 12809-12813(October 1998). There remains, however, a need for an engineered pNBesterase that provides selectivity and high yields in the deprotectionof pNB-protected carbapenem intermediates, such as the pNB-protectedimipenem of compound (2), under commercially viable and industriallyuseful process conditions.

4. SUMMARY

The present disclosure provides engineered polypeptides having pNBesterase activity, polynucleotides encoding the polypeptides, methods ofthe making the polypeptides, and methods of using the polypeptides forthe selective removal of pNB protecting groups in the synthesis ofcarbapenem products, such as imipenem. The engineered polypeptideshaving pNB esterase activity of the present disclosure have beenengineered to have one or more residue differences as compared to thewild-type pNB esterase. They also have one or more residue differencesas compared to the previously engineered pNB esterase polypeptide ofamino acid sequence SEQ ID NO: 2, which has enhanced solvent and thermalstability relative to the wild-type pNB esterase of Bacillus subtilis.In particular, the engineered pNB esterase polypeptides of the presentdisclosure have been engineered for efficient removal a pNB protectinggroup from the imipenem precursor compound of compound (2) therebyconverting it to the product compound (1), imipenem, as shown in Scheme1.

The amino residue differences are located at residue positions affectingvarious enzyme properties, including among others, activity, stability,product selectivity, and product tolerance. These amino residuedifferences, although evolved for the removal of pNB protecting groupsfrom the imipenem precursor of compound (2), can also be used to evolveengineered pNB esterase polypeptides having activity useful for removingpNB protecting groups of structurally analogous carbapenem precursorcompounds including, but not limited to, pNB-protected precursorcompounds of thienamycin meropenem, doripenem, ertapenem, biopenem, andpanipenem.

In one aspect, the present disclosure provides engineered polypeptideshaving pNB esterase activity, where the engineered polypeptide comprisesan amino acid sequence having at least 80% identity to SEQ ID NO: 2 andone or more residue differences as compared to SEQ ID NO:2 at residuepositions selected from: X108, X115, X116, X130, X193, X214, X219, X273,X276, X321, and X362. In some embodiments, the residue differences ascompared to SEQ ID NO: 2 at the residue positions X108, X115, X116,X130, X193, X214, X219, X273, X276, X321, and X362 are selected fromX108L/Y, X115Q/W, X116S, X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V,X273A/E/T/V, X276A/T/L, X321A, and X362A/D/Q/S/V.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2 and one or more residue differences as compared to SEQID NO: 2 selected from: X108L/Y, X193A/D/E/V, X219A/D/L/V, X273A/E/T/V,and X362A/D/Q/S/V.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2 and a residue difference as compared to SEQ ID NO: 2 atposition X193 selected from: X193A/D/E/V. In some embodiments, the aminoacid residue difference as compared to SEQ ID NO: 2 at position X193 isX193V.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2, and an amino acid difference as compared to SEQ ID NO:2 of X193V, and further comprises residue differences as compared to SEQID NO: 2 at positions X219 and X273 selected from X219L/V and X273A/V.In further embodiments, the amino acid sequence further comprisesresidue differences as compared to SEQ ID NO: 2 at positions X108 andX362 selected from X108L/Y and X362A/D/Q/S/V. In still furtherembodiments, the engineered polypeptide further comprises a residuedifference as compared to SEQ ID NO: 2 at position X115 selected fromX115Q/W.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2, and a combination of residue differences as compared toSEQ ID NO: 2 selected from: (a) X193V, X219V, and X273A; (b) X108Y,X193D, X219V, X273A, and X362S; (c) X108Y, X193V, X219V, X273A, andX362Q; (d) X108Y, X115Q, X193V, X219L, X273A, and X362Q; and (e) X108Y,X115Q, X193V, X219V, X273A, and X362Q.

In some embodiments, the engineered polypeptides having pNB esteraseactivity disclosed above (and elsewhere herein) can have additionalresidue differences at other residue positions. In some embodiments, theengineered pNB esterases can have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40,1-45, or 1-50 additional residue differences as compared to SEQ ID NO:2.In some embodiments, the engineered pNB esterases can have 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 30, 35, 40, 45, or 50 additional residue differences. Insome embodiments, the amino acid sequence has additionally 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25residue differences as compared to SEQ ID NO: 2.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2, one or more amino acid residue difference as comparedto SEQ ID NO: 2 selected from X108L/Y, X115Q/W, X193A/D/E/V,X219A/D/L/V, X273A/E/T/V, and X362A/D/Q/S/V, and the amino acid sequencefurther comprises one or more residue differences as compared to SEQ IDNO: 2 selected from: X116S, X130T, X164T, X214G, X276A/T/L, and X321A.In still further embodiments, the amino acid sequence can furthercomprise a residue difference as compared to SEQ ID NO: 2 selected from:X49G, X94G, X96S, X227T, X251V, X267R, X271L, X274L, X313F, X322C/Y,X343V, X356R, X359A, X398L, X412E, X437T, X464A, and X481R.

In some embodiments, the engineered polypeptide having pNB-esteraseactivity comprises an amino acid sequence having at least 80% identityto SEQ ID NO: 2, and of any of the amino acid differences as compared toSEQ ID NO: 2 as disclosed herein (e.g., as disclosed in the exemplarypolypeptides of Table 2), but in which the amino acid sequence does notcomprise a residue difference as compared to SEQ ID NO: 2 at positionsX60, X144, X317, X322, X334, X358, and X370.

In some embodiments, the engineered polypeptides having pNB esteraseactivity of the present disclosure having at least 80% identity to SEQID NO: 2 and any of the amino acid residue differences disclosed herein,further comprises at least 1.2 fold, 2 fold, 5 fold, 10 fold, 20 fold,25 fold, 50 fold, 75 fold, 100 fold, or greater increased activity ascompared to the polypeptide of SEQ ID NO: 4 in converting compound (2)to compound (1) under suitable reaction conditions.

In some embodiments, the engineered polypeptides having pNB esteraseactivity comprises an amino acid sequence having at least 80%, 85%, 87%,88%, 89%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to a sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,and 120.

In some embodiments, that disclosure further provides any of theengineered polypeptides having pNB esterase activity as disclosedherein, wherein the polypeptide is immobilized on a solid support. Insome embodiments, the solid support is a bead or resin comprisingpolymethacrylate with epoxide functional groups, polymethacrylate withamino epoxide functional groups, styrene/DVB copolymer orpolymethacrylate with octadecyl functional groups

Exemplary engineered polypeptides having pNB esterase activity and aminoacid sequences incorporating the residue differences disclosed herein,including various combinations thereof, and having improved properties(e.g., capable of converting compound (2) to compound (1) under suitablereaction conditions) are disclosed in Table 2, and the Examples. Theamino acid sequences are provided in the Sequence Listing and includeSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and 120.

In another aspect, the present disclosure provides polynucleotidesencoding the engineered polypeptides having pNB esterase activity, aswell as expression vectors comprising the polynucleotides, and hostcells capable of expressing the polynucleotides encoding the engineeredpolypeptides. Exemplary polynucleotide sequences are provided in theSequence Listing incorporated by reference herein and include SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, and 119.

In some embodiments, the present disclosure also provides methods ofmanufacturing the engineered polypeptides having pNB esterase activity,where the method can comprise culturing a host cell capable ofexpressing a polynucleotide encoding the engineered polypeptide underconditions suitable for expression of the polypeptide. In someembodiments, the method for manufacturing the engineered pNB esterasepolypeptide can also include: (a) synthesizing a polynucleotide encodinga polypeptide comprising an amino acid sequence selected from SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, and 120, and having one or more residuedifferences as compared to SEQ ID NO:2 at residue positions X108, X115,X116, X130, X193, X214, X219, X273, X276, X321, and X362 selected fromX108L/Y, X115Q/W, X116S, X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V,X273A/E/T/V, X276A/T/L, X321A, and X362A/D/Q/S/V; and (b) expressing thepNB esterase polypeptide encoded by the polynucleotide. As furtherprovided in the detailed description, additional variations can beincorporated during the synthesis of the polynucleotide to prepareengineered polypeptides with corresponding differences in the expressedamino acid sequences.

The structural features of the engineered pNB esterase polypeptidesallow for the conversion of the pNB-protected substrate of compound (2)to their corresponding un-protected product of compound (1), imipenem.Thus, in another aspect the present disclosure provides a process forpreparing carbapenem antibiotic of compound (1), imipenem, or a salt orhydrate of compound (1),

wherein the method comprises contacting a substrate compound (2), or asalt of hydrate of compound (2),

with an engineered pNB esterase polypeptide of the present disclosure,under suitable reaction conditions. As provided herein, the processesusing the engineered pNB esterases can be carried out under a range ofsuitable reaction conditions, including, among others, pH, temperature,buffer, solvent system, substrate loading, polypeptide loading, cofactorloading, pressure, and reaction time. In some embodiments, the suitablereaction conditions for a biocatalytic process using the engineered pNBesterases of the present disclosure can comprise: (a) substrate loadingat about 2 g/L to 200 g/L; (b) about 0.1 to 10 g/L of engineered pNBesterase polypeptide; (c) about 0.05 to 0.5 M MES buffer; (d) about 5%to about 20% (v/v) DMF co-solvent; (e) pH of about 6 to 8; and/or (f)temperature of about 10 to 35° C. Further, guidance on the choice ofengineered pNB esterases, preparation of the biocatalysts, andparameters and reaction conditions for carrying out the processes aredescribed in the detailed description that follow.

5. DETAILED DESCRIPTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contextclearly indicates otherwise. Thus, for example, reference to “apolypeptide” includes more than one polypeptide. Similarly, “comprise,”“comprises,” “comprising” “include,” “includes,” and “including” areinterchangeable and not intended to be limiting. It is to be furtherunderstood that where descriptions of various embodiments use the term“comprising,” those skilled in the art would understand that in someinstances, the embodiment can be alternatively described using the terms“consisting essentially of” or “consisting of.”

It is to be understood that both the foregoing general description, andthe following detailed description are exemplary and explanatory onlyand are not restrictive of this disclosure. The section headings usedherein are for organizational purposes only and not to be construed aslimiting the subject matter described.

5.1 Abbreviations

The abbreviations used for the genetically encoded amino acids areconventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIS H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (C_(α)). For example, whereas “Ala”designates alanine without specifying the configuration about theα-carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the α-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When polypeptide sequences are presented as astring of one-letter or three-letter abbreviations (or mixturesthereof), the sequences are presented in the amino (N) to carboxy (C)direction in accordance with common convention.

The abbreviations used for the genetically encoding nucleosides areconventional and are as follows: adenosine (A); guanosine (G); cytidine(C); thymidine (T); and uridine (U). Unless specifically delineated, theabbreviated nucleotides may be either ribonucleosides or2′-deoxyribonucleosides. The nucleosides may be specified as beingeither ribonucleosides or 2′-deoxyribonucleosides on an individual basisor on an aggregate basis. When nucleic acid sequences are presented as astring of one-letter abbreviations, the sequences are presented in the5′ to 3′ direction in accordance with common convention, and thephosphates are not indicated.

5.2 Definitions

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings.

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Polynucleotide” or “nucleic acid” refers to two or more nucleosidesthat are covalently linked together. The polynucleotide may be whollycomprised ribonucleosides (i.e., an RNA), wholly comprised of2′-deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and2′-deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine and cytosine), itmay include one or more modified and/or synthetic nucleobases, such as,for example, inosine, xanthine, hypoxanthine, etc. Preferably, suchmodified or synthetic nucleobases will be encoding nucleobases.

“pNB esterase activity” as used herein refers to the enzymatic activityof hydrolyzing a para-nitrobenzyl ester group to form para-nitrophenoland the corresponding acid of the ester.

“pNB esterase” as used herein refers to an enzyme having pNB esteraseactivity and can include a naturally occurring (wild-type) pNB esterase,such as the pNB esterase from Bacillus subtilis, as well asnon-naturally occurring engineered pNB esterase polypeptides generatedby human manipulation.

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally-occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” or “engineered” or “non-naturally occurring” when usedwith reference to, e.g., a cell, nucleic acid, or polypeptide, refers toa material, or a material corresponding to the natural or native form ofthe material, that has been modified in a manner that would nototherwise exist in nature, or is identical thereto but produced orderived from synthetic materials and/or by manipulation usingrecombinant techniques. Non-limiting examples include, among others,recombinant cells expressing genes that are not found within the native(non-recombinant) form of the cell or express native genes that areotherwise expressed at a different level.

“Percentage of sequence identity” and “percentage homology” are usedinterchangeably herein to refer to comparisons among polynucleotides andpolypeptides, and are determined by comparing two optimally alignedsequences over a comparison window, wherein the portion of thepolynucleotide or polypeptide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence for optimal alignment of the two sequences. Thepercentage may be calculated by determining the number of positions atwhich the identical nucleic acid base or amino acid residue occurs inboth sequences to yield the number of matched positions, dividing thenumber of matched positions by the total number of positions in thewindow of comparison and multiplying the result by 100 to yield thepercentage of sequence identity. Alternatively, the percentage may becalculated by determining the number of positions at which either theidentical nucleic acid base or amino acid residue occurs in bothsequences or a nucleic acid base or amino acid residue is aligned with agap to yield the number of matched positions, dividing the number ofmatched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Those of skill in the art appreciate that there aremany established algorithms available to align two sequences. Optimalalignment of sequences for comparison can be conducted, e.g., by thelocal homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math.2:482, by the homology alignment algorithm of Needleman and Wunsch,1970, J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package), or by visualinspection (see generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (1995Supplement) (Ausubel)). Examples of algorithms that are suitable fordetermining percent sequence identity and sequence similarity are theBLAST and BLAST 2.0 algorithms, which are described in Altschul et al.,1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, NucleicAcids Res. 3389-3402, respectively. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information website. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as, theneighborhood word score threshold (Altschul et al, supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are then extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4, and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlength(W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915).Exemplary determination of sequence alignment and % sequence identitycan employ the BESTFIT or GAP programs in the GCG Wisconsin Softwarepackage (Accelrys, Madison Wis.), using default parameters provided.

“Reference sequence” refers to a defined sequence used as a basis for asequence comparison. A reference sequence may be a subset of a largersequence, for example, a segment of a full-length gene or polypeptidesequence. Generally, a reference sequence is at least 20 nucleotide oramino acid residues in length, at least 25 residues in length, at least50 residues in length, or the full length of the nucleic acid orpolypeptide. Since two polynucleotides or polypeptides may each (1)comprise a sequence (i.e., a portion of the complete sequence) that issimilar between the two sequences, and (2) may further comprise asequence that is divergent between the two sequences, sequencecomparisons between two (or more) polynucleotides or polypeptide aretypically performed by comparing sequences of the two polynucleotides orpolypeptides over a “comparison window” to identify and compare localregions of sequence similarity. In some embodiments, a “referencesequence” can be based on a primary amino acid sequence, where thereference sequence is a sequence that can have one or more changes inthe primary sequence. For instance, a “reference sequence based on SEQID NO:2 having at the residue corresponding to X193 an alanine” or“X193A” refers to a reference sequence of SEQ ID NO:2 in which thecorresponding residue at X193 (which is a methionine in SEQ ID NO:2),has been changed to alanine.

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequencefor optimal alignment of the two sequences. The comparison window can belonger than 20 contiguous residues, and includes, optionally 30, 40, 50,100, or longer windows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent identity and 89 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 residue positions, frequentlyover a window of at least 30-50 residues, wherein the percentage ofsequence identity is calculated by comparing the reference sequence to asequence that includes deletions or additions which total 20 percent orless of the reference sequence over the window of comparison. Inspecific embodiments applied to polypeptides, the term “substantialidentity” means that two polypeptide sequences, when optimally aligned,such as by the programs GAP or BESTFIT using default gap weights, shareat least 80 percent sequence identity, preferably at least 89 percentsequence identity, at least 95 percent sequence identity or more (e.g.,99 percent sequence identity). Preferably, residue positions which arenot identical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredpNB esterase, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Amino acid difference” or “residue difference” refers to a change inthe amino acid residue at a position of a polypeptide sequence relativeto the amino acid residue at a corresponding position in a referencesequence. The positions of amino acid differences generally are referredto herein as “Xn,” where n refers to the corresponding position in thereference sequence upon which the residue difference is based. Forexample, a “residue difference at position X193 as compared to SEQ IDNO: 2” refers to a change of the amino acid residue at the polypeptideposition corresponding to position 193 of SEQ ID NO:2. Thus, if thereference polypeptide of SEQ ID NO: 2 has a methionine at position 193,then a “residue difference at position X193 as compared to SEQ ID NO:2”an amino acid substitution of any residue other than methionine at theposition of the polypeptide corresponding to position 193 of SEQ ID NO:2. In most instances herein, the specific amino acid residue differenceat a position is indicated as “XnY” where “Xn” specified thecorresponding position as described above, and “Y” is the single letteridentifier of the amino acid found in the engineered polypeptide (i.e.,the different residue than in the reference polypeptide). In someembodiments, where more than one amino acid can appear in a specifiedresidue position, the alternative amino acids can be listed in the formXnY/Z, where Y and Z represent alternate amino acid residues. In someinstances (e.g., in Table 2), the present disclosure also providesspecific amino acid differences denoted by the conventional notation“AnB”, where A is the single letter identifier of the residue in thereference sequence, “n” is the number of the residue position in thereference sequence, and B is the single letter identifier of the residuesubstitution in the sequence of the engineered polypeptide. Furthermore,in some instances, a polypeptide of the present disclosure can includeone or more amino acid residue differences relative to a referencesequence, which is indicated by a list of the specified positions wherechanges are made relative to the reference sequence.

“Conservative amino acid substitution” refers to a substitution of aresidue with a different residue having a similar side chain, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. By way of example and not limitation, an amino acid with analiphatic side chain may be substituted with another aliphatic aminoacid, e.g., alanine, valine, leucine, and isoleucine; an amino acid withhydroxyl side chain is substituted with another amino acid with ahydroxyl side chain, e.g., serine and threonine; an amino acid havingaromatic side chains is substituted with another amino acid having anaromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, andhistidine; an amino acid with a basic side chain is substituted withanother amino acid with a basic side chain, e.g., lysine and arginine;an amino acid with an acidic side chain is substituted with anotheramino acid with an acidic side chain, e.g., aspartic acid or glutamicacid; and a hydrophobic or hydrophilic amino acid is replaced withanother hydrophobic or hydrophilic amino acid, respectively. Exemplaryconservative substitutions are provided in Table 1 below.

TABLE 1 Residue Possible Conservative Substitutions A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) N, Q, S, T Other polar H, Y, W, F Other aromatic (H, Y, W, F) C,P None

“Non-conservative substitution” refers to substitution of an amino acidin the polypeptide with an amino acid with significantly differing sidechain properties. Non-conservative substitutions may use amino acidsbetween, rather than within, the defined groups and affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine), (b) the charge or hydrophobicity, or (c) the bulkof the side chain. By way of example and not limitation, an exemplarynon-conservative substitution can be an acidic amino acid substitutedwith a basic or aliphatic amino acid; an aromatic amino acid substitutedwith a small amino acid; and a hydrophilic amino acid substituted with ahydrophobic amino acid.

“Deletion” refers to modification to the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, or upto 20% of the total number of amino acids making up the reference enzymewhile retaining enzymatic activity and/or retaining the improvedproperties of an engineered pNB esterase enzyme. Deletions can bedirected to the internal portions and/or terminal portions of thepolypeptide. In various embodiments, the deletion can comprise acontinuous segment or can be discontinuous.

“Insertion” refers to modification to the polypeptide by addition of oneor more amino acids from the reference polypeptide. In some embodiments,the improved engineered pNB esterase enzymes comprise insertions of oneor more amino acids to the naturally occurring pNB esterase polypeptideas well as insertions of one or more amino acids to other improved pNBesterase polypeptides. Insertions can be in the internal portions of thepolypeptide, or to the carboxy or amino terminus. Insertions as usedherein include fusion proteins as is known in the art. The insertion canbe a contiguous segment of amino acids or separated by one or more ofthe amino acids in the reference polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length pNB esterase polypeptide,for example the reference engineered pNB esterase polypeptide of SEQ IDNO: 2.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved pNB esterase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved pNB esterase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis, it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure pNB esterase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved pNB esterase polypeptide is a substantially pure polypeptidecomposition.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (e.e.) calculated therefromaccording to the formula [major enantiomer−minor enantiomer]/[majorenantiomer+minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereoselectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Improved enzyme property” refers to a pNB esterase polypeptide thatexhibits an improvement in any enzyme property as compared to areference pNB esterase. For the engineered pNB esterase polypeptidesdescribed herein, the comparison is generally made to the referenceengineered pNB esterase enzyme of SEQ ID NO: 4, although in someembodiments, the reference pNB esterase can be another engineered pNBesterase, or the wild-type pNB esterase of B. subtilis. Enzymeproperties for which improvement is desirable include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate), thermostability, solventstability, product selectivity, pH activity profile, refractoriness toinhibitors (e.g., substrate or product inhibition), andstereoselectivity (including enantioselectivity).

“Increased enzymatic activity” refers to an improved property of theengineered pNB esterase polypeptides, which can be represented by anincreased specific activity (e.g., product produced/time/weight protein)or an increased percent conversion of the substrate to the product(e.g., percent conversion of starting amount of substrate to product ina specified time period using a specified amount of pNB esterase) ascompared to the reference pNB esterase enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.2 fold the enzymatic activity of the correspondingwild-type pNB esterase enzyme, to as much as 2 fold, 5 fold, 10 fold, 20fold, 25 fold, 50 fold, 75 fold, 100 fold, or more enzymatic activitythan the naturally occurring pNB esterase or another engineered pNBesterase from which the pNB esterase polypeptides were derived. PNBesterase activity can be measured by any one of standard assays, such asby monitoring changes in spectrophotometric properties of reactants orproducts. In some embodiments, the amount of products produced can bemeasured by High-Performance Liquid Chromatography (HPLC) separationcombined with UV absorbance or fluorescent detection followingderivatization, such as with o-phthaldialdehyde (OPA). Comparisons ofenzyme activities are made using a defined preparation of enzyme, adefined assay under a set condition, and one or more defined substrates,as further described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

“Conversion” refers to the enzymatic conversion of the substrate(s) tothe corresponding product(s). “Percent conversion” refers to the percentof the substrate that is converted to the product within a period oftime under specified conditions. Thus, the “enzymatic activity” or“activity” of a pNB esterase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” refers to a pNB esterase polypeptide that maintainssimilar activity (more than 60% to 80% for example) after exposure toelevated temperatures (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the wild-type enzyme.

“Solvent stable” refers to a pNB esterase polypeptide that maintainssimilar activity (more than e.g., 60% to 80%) after exposure to varyingconcentrations (e.g., 5-99%) of solvent (ethanol, isopropyl alcohol,dimethylsulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran,acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for aperiod of time (e.g., 0.5-24 hrs) compared to the wild-type enzyme.

“Thermo- and solvent stable” refers to a pNB esterase polypeptide thatis both thermostable and solvent stable.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci. USA 83:8893-8897; Freier et al., 1986, Proc. Natl.Acad. Sci. USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846;Rychlik et al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991,Nucleic Acids Res 19:698); Sambrook et al., supra); Suggs et al., 1981,In Developmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporated herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered pNB esterase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA, with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thepNB esterase enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the nucleic acid sequence encoding thepolypeptide. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleic acid sequence encoding a polypeptide.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” refers to a nucleic acid sequence that is recognizedby a host cell for expression of a polynucleotide of interest, such as acoding sequence. The promoter sequence contains transcriptional controlsequences, which mediate the expression of a polynucleotide of interest.The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

“Suitable reaction conditions” refer to those conditions in thebiocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a pNB esterase polypeptide of the presentdisclosure is capable of converting a substrate compound to a productcompound (e.g., conversion of compound (2) to compound (1)). Exemplary“suitable reaction conditions” are provided in the detailed descriptionand illustrated by the Examples.

“Loading”, such as in “compound loading” or “enzyme loading” or“cofactor loading” refers to the concentration or amount of a componentin a reaction mixture at the start of the reaction.

“Substrate” in the context of a biocatalyst mediated process refers tothe compound or molecule acted on by the biocatalyst. For example, anexemplary substrate for the engineered pNB esterase biocatalysts in theprocess disclosed herein is compound (2).

“Product” in the context of a biocatalyst mediated process refers to thecompound or molecule resulting from the action of the biocatalyst. Forexample, an exemplary product for the engineered pNB esterasebiocatalysts in the process disclosed herein is compound (1).

“Protecting group” refers to a group of atoms that mask, reduce orprevent the reactivity of the functional group when attached to areactive functional group in a molecule. Typically, a protecting groupmay be selectively removed as desired during the course of a synthesis.Examples of protecting groups can be found in Wuts and Greene, “Greene'sProtective Groups in Organic Synthesis,” 4^(th) Ed., Wiley Interscience(2006), and Harrison et al., Compendium of Synthetic Organic Methods,Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Functional groups that canhave a protecting group include, but are not limited to, hydroxy, amino,and carboxy groups.

5.3 Engineered pNB Esterase Polypeptides

The present disclosure provides engineered polypeptides having pNBesterase activity, polynucleotides encoding the polypeptides, andmethods for using the polypeptides. Where the foregoing descriptionrelates to polypeptides, it is to be understood that it also describesthe polynucleotides encoding the polypeptides.

The present disclosure relates to engineered pNB esterase polypeptidesderived from the wild-type pNB esterase polypeptide of Bacillus subtilisof GenBank Access. No.: AAA81915.1, GI:468046. The engineered pNBesterases of the present disclosure have been engineered with amino acidresidue substitutions that allow for conversion of a pNB-protectedsubstrate of compound (2) to the corresponding deprotected product ofcompound (1), imipenem. Significantly, the present disclosure identifiesamino acid residue positions and corresponding amino acid residuesubstitutions in the engineered pNB esterase polypeptide that canincrease the enzymatic activity, product selectivity, and stability, indeprotecting pNB-protected carbapenem substrates.

The identification of the specific residue positions and substitutionsin the engineered pNB esterase polypeptides of the present disclosure byengineering through directed evolution methods using structure-basedrational sequence library design with screening for improved functionalproperties using an activity assay based on the conversion of thepNB-protected imipenem precursor of compound (2) to its correspondingunprotected product of compound (1), imipenem. Specifically, theconversion of substrate compound (2) to product compound (1) as shown inScheme 1 (above). The engineered pNB esterase polypeptides of thepresent disclosure were evolved to efficiently convert the pNB-protectedimipenem substrate of compound (2) to the product compound (1), undersuitable reaction conditions.

The specific structural features and structure-function correlatinginformation of the engineered pNB esterase polypeptides of the presentdisclosure also allow for the rational design and directed evolution ofengineered pNB esterase polypeptides that can carry out the selectivedeprotection of other pNB-protected carbapenem compounds (other thancompound (2)), to the corresponding carbapenem product compound (otherthan compound (1)). In some embodiments, the engineered pNB esterasepolypeptides of the present disclosure are capable of convertingpNB-protected carbapenem compounds which are structural analogs ofcompound (2), to their corresponding deprotected carbapenem productcompounds which are structural analogs of compound (1).

The engineered pNB esterase polypeptides adapted for efficientconversion of compound (2) to compound (1) have one or more residuedifferences as compared to the amino acid sequence of the referenceengineered pNB esterase polypeptide of SEQ ID NO: 2. The residuedifferences are associated with enhancements in enzyme properties,including enzymatic activity, enzyme stability, and resistance toformation of undesirable side-products, such as a β-lactam ring-openeddiacid of compound (3).

In some embodiments, the engineered pNB esterase polypeptides showincreased activity in the conversion of pNB-protected substrate compound(2) to the deprotected product compound (1) in a defined time with thesame amount of enzyme as compared to the reference engineered pNBesterase of SEQ ID NO: 4. In some embodiments, the engineered pNBesterase polypeptide has at least about 1.2 fold, 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, or 50 fold ormore the activity as compared to the reference engineered polypeptiderepresented by SEQ ID NO:4 under suitable reaction conditions.

In some embodiments, the engineered pNB esterase polypeptides arecapable of converting substrate compound (2) to product compound (1)with increased tolerance for the presence of substrate relative to thereference polypeptide of SEQ ID NO: 4 under suitable reactionconditions. Thus, in some embodiments the engineered pNB esterasepolypeptides are capable of converting the substrate compound (2) toproduct compound (1) under a substrate loading concentration of at leastabout 1 g/L, about 5 g/L, about 10 g/L, about 20 g/L, about 30 g/L,about 40 g/L, about 50 g/L, about 70 g/L, about 100 g/L, about 125 g/L,about 150 g/L. about 175 g/L or about 200 g/L or more with a percentconversion of at least about at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 98%, or at least about 99%, in a reaction time ofabout 72 h or less, about 48 h or less, about 36 h or less, or about 24h less, under suitable reaction conditions.

In some embodiments, the engineered pNB esterase polypeptides arecapable of converting at least 70% of substrate compound (2) to productcompound (1) in 2 h, at a substrate loading of 25 g/L and a temperatureof 15° C.

In some embodiments, the engineered pNB esterase polypeptides arecapable of converting substrate compound (2) to product compound (1)with a selectivity ratio for product compound (1) to side-productcompound (3) of at least 50:1, at least 60:1, at least 70:1, at least80:1, at least 90:1, or greater.

The suitable reaction conditions under which the above-describedimproved properties of the engineered polypeptides carry out theconversion can be determined with respect to concentrations or amountsof polypeptide, substrate, buffer, co-solvent, pH, and/or conditionsincluding temperature and reaction time, as further described below andin the Examples.

The present disclosure provides 59 exemplary engineered pNB esterasepolypeptides having structural features capable of converting thesubstrate of compound (2), a pNB-protected precursor of imipenem, to thecorresponding product of compound (1), imipenem. The present disclosureprovides the sequence structure of the 59 exemplary engineered pNBesterase polypeptides as SEQ ID NOs: 3-120 in the electronic SequenceListing file accompanying this disclosure, which is hereby incorporatedby reference herein. The odd numbered sequence identifiers (i.e., SEQ IDNOs) refer to the nucleotide sequence encoding the amino acid sequenceprovided by the even numbered SEQ ID NOs. The present disclosure alsoprovides in Table 2 sequence structural information correlating specificamino acid sequence features with the functional activity of theengineered pNB esterase polypeptides. This structure-functioncorrelation information is provided in the form of specific amino acidresidues differences relative to the reference engineered polypeptide ofSEQ ID NO: 2 and associated experimentally determined activity data forthe 59 exemplary engineered pNB esterases of SEQ ID NOs: 3-120. Theamino acid residue differences are based on comparison to the referencesequence of SEQ ID NO: 2, which has the following 7 amino acid residuedifferences relative to the sequence of the wild-type pNB-esterase fromBacillus subtilis (GenBank Access. No.: AAA81915.1, GI:468046): I60V,L144M, P317S, H322R, L334S, M358V, and Y370F. As noted in Table 2, theengineered polypeptide of SEQ ID NO: 2 does not have detectable activityin the conversion of compound (2) to compound (1). The engineered pNBesterase polypeptide of SEQ ID NO: 4 which has the single amino aciddifference M193A as compared to SEQ ID NO: 2 was found to havedetectable activity in the conversion of compound (2) to compound (1).It was used as the reference for the relative activity measurements. Therelative pNB esterase activity of each exemplary engineered pNB esterasepolypeptide was determined as conversion of the substrate compound (2)to the imipenem product of compound (1) in comparison to the pNBesterase activity of the engineered pNB esterase polypeptide of SEQ IDNO: 4 over a set time period and temperature in a high-throughput (HTP)assay. The Activity Improvement values in Table 2 were determined usingan assay of E. coli clear cell lysates in 96 well-plate format of ˜250μL volume per well following assay reaction conditions as noted in thetable and the Examples.

TABLE 2 Engineered pNB Esterase Polypeptides and Relative ActivityImprovement Activity SEQ ID Improvement NO: Amino Acid Differences(relative to (nt/aa) (relative to SEQ ID NO: 2) SEQ ID NO: 4) 1/2 Nonenot detected 3/4 M193A; 1 5/6 F108Y; P116S; M193A; 1.4 7/8 F108Y; M193A;1.8  9/10 E115W; M193A; 1.0 11/12 E115Q; M193A; 1.2 13/14 M193V; E214G;4.3 15/16 M193A; R219D; 9.5 17/18 M193A; R219L; 3.8 19/20 M193A; L273E;12.1 21/22 M193A; Q276H; 10.4 23/24 M193A; Q276T; 3.2 25/26 M193A;Q276L; 7.9 27/28 I130T; M193A; Q276H; 10.4 29/30 M193A; L362D; 13.931/32 M193A; L362S; 1.6 33/34 M193A; L362Q; 6.9 35/36 M193E; 48.1 37/38M193D; 7.2 39/40 F108L; M193V; 7.4 41/42 M193V; L273V; Q276A; 15.7 43/44M193V; R219V; L273V; Q276A; L362V; 8.9 45/46 F108L; M193A; R219V; L273V;19.9 47/48 F108L; R219V; L273V; 10.0 49/50 M193V; L273V; Q276A; L362A;14.0 51/52 M193V; R219A; L273V; L362V; 22.0 53/54 M193V; R219V; L273A;47.2 55/56 M193V; L273A; Q276A; V321A; L362A; 13.5 57/58 M193V; R219A;L273A; Q276A; L362V; 9.9 59/60 F108L; R219V; L362A; 9.6 61/62 F108L;R219V; L362V; 14.0 63/64 F108L; M193V; L273A; L362A; 16.1 65/66 M193V;R219V; L273A; Q276A; L362A; 20.5 67/68 M193V; R219V; L273A; L362V; 13.269/70 M193V; L273V; 20.5 71/72 F108L; M193V; R219A; L273A; L362A; 13.073/74 A164T; M193V; R219V; L273V; 9.2 Q276A; L362V; 75/76 F108Y; E115Q;M193V; R219V; 247 L273A; L362Q; 77/78 M193V; R219V; Q276L; L362Q; 11979/80 F108Y; E115Q; M193V; R219L; 239 L273A; L362Q; 81/82 F108Y; E115Q;M193D; R219V; L273A; 143 83/84 F108Y; M193D; R219D; Q276L; 76 85/86F108Y; M193E; R219D; 47 87/88 F108Y; M193D; R219V; L273A; 220 89/90F108Y; E115Q; M193D; R219V; 204 L273A; Q276L; L362S; 91/92 F108Y; M193V;R219V; L273A; 122 93/94 F108Y; M193D; R219V; L273A; L362S; 205 95/96F108Y; E115Q; M193D; R219L; 43 L273A; L362S; 97/98 F108Y; E115Q; M193D;R219V; L273A; 174  99/100 F108Y; E115W; M193D; R219L; 92 L273A Q276L;L362Q; 101/102 E115Q; M193V; R219V; L273A; L362Q; 172 103/104 F108Y;E115W; M193D; R219L; 152 105/106 F108Y; M193D; R219V; L273A; 174 107/108E115Q; M193D; R219L; L273A; Q276L; 161 L362D; 109/110 F108Y; M193D;R219L; L273A; L362Q; 185 111/112 F108Y; M193V; R219V; L273A; L362Q; 186113/114 E115W; M193D; R219V; L273A; 186 115/116 F108Y; M193D; R219L;L273A; L362S; 232 117/118 F108Y; M193D; R219L; L273A 186 119/120 F108Y;E115Q; M193D; R219L; L273A; 166 Q276L; ¹Activity Improvement (relativeto SEQ ID NO: 4) is calculated as the ratio of % conversion of productformed by the engineered pNB-esterase polypeptide of interest to the %conversion of the reference polypeptide of SEQ ID NO: 4 under ReactionConditions A. % Conversion was quantified by dividing the areas of theproduct peak by the sum of the areas of the substrate and product peakas determined by HPLC analysis. Reaction Conditions A: 2 g/L substrateof compound (2), 125 μL lysate (prepared by adding 200 μL of LysisBuffer (1 mg/mL lysozyme, 0.5 mg/mL polymyxin B sulfate, 0.1M phosphatebuffer, pH 7.5) to E. coli expressing polypeptide of interest grown in96 well plates), 0.1M phosphate buffer, pH 7.5, 15% (v/v) DMF, 15° C., 2h. Total reaction volume is 200 μL.

From an inspection of the amino acid sequences, and results for the 59exemplary engineered pNB esterase polypeptides of Table 2, improvedproperties of increased activity, selectivity, and/or stability, thatare associated with one or more residue differences as compared to SEQID NO: 2 at the following residue positions: X108, X115, X116, X130,X193, X214, X219, X273, X276, X321, and X362. The specific amino aciddifferences as compared to SEQ ID NO: 2 at each of these positions thatare associated with the improved properties include X108L/Y, X115Q/W,X116S, X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V,X276A/T/L, X321A, and X362A/D/Q/S/V.

In some embodiments, the engineered pNB esterase polypeptides of thepresent disclosure comprise amino acid sequences having residuedifferences as compared to the engineered pNB esterase represented bySEQ ID NO:2 at residue positions selected from: X108, X115, X116, X130,X193, X214, X219, X273, X276, X321, and X362. In some embodiments, thespecific amino acid residue differences as compared to SEQ ID NO: 2 atresidue positions X108, X115, X116, X130, X193, X214, X219, X273, X276,X321, and X362, are selected from: X108L/Y, X115Q/W, X116S, X130T,X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L, X321A,and X362A/D/Q/S/V.

As will be appreciated by the skilled artisan, residue differencesdisclosed in Table 2 have no significant deleterious effects on pNBesterase activity and/or product selectivity for the engineered pNBesterase polypeptides, all of which maintain pNB esterase activity forthe conversion of compound (2) to compound (1). Accordingly, the skilledartisan will understand that the residue differences at the residuepositions disclosed herein can be used individually or in variouscombinations to produce engineered pNB esterase polypeptides having thedesired functional properties, including, among others, pNB esteraseactivity, selectivity, and stability, in converting pNB-protectedcarbapenem compounds, such as compound (2) and its structural analogs,to its corresponding deprotected carbapenem compound, such as compound(1), imipenem.

In light of the guidance provided herein, it is further contemplatedthat any of the exemplary engineered polypeptides of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, and 120 can be used as the starting amino acid sequencefor synthesizing other engineered pNB esterase polypeptides, for exampleby subsequent rounds of evolution by adding new combinations of variousamino acid differences from other polypeptides in Table 2, and otherresidue positions described herein. Further improvements may begenerated by including amino acid differences at residue positions thathad been maintained as unchanged throughout earlier rounds of evolution.

Accordingly, in some embodiments, the present disclosure providesengineered polypeptides having pNB esterase activity, and optionallyimproved properties in converting a pNB-protected substrate of compound(2) to a deprotected product compound (1) as compared to a referencepolypeptide of SEQ ID NO:4, wherein the polypeptide comprises an aminoacid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity toreference sequence SEQ ID NO: 2 and one or more residue differences ascompared to SEQ ID NO:2 at residue positions selected from X108, X115,X116, X130, X193, X214, X219, X273, X276, X321, and X362. In someembodiments, the specific amino acid residue differences as compared toSEQ ID NO: 2 at residue positions X108, X115, X116, X130, X193, X214,X219, X273, X276, X321, and X362, are selected from: X108L/Y, X115Q/W,X116S, X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V,X276A/T/L, X321A, and X362A/D/Q/S/V.

In some embodiments, the present disclosure provides an engineeredpolypeptide having pNB esterase activity that comprises an amino acidsequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to referencesequence SEQ ID NO:2 and one or more residue differences as compared toSEQ ID NO: 2 at residue positions selected from as compared to SEQ IDNO:2 at residue positions X108, X115, X193, X219, X273, X276, and X362are selected from: X108L/Y, X115Q/W, X193A/D/E/V, X219A/D/L/V,X273A/E/T/V, X276A/T/L, and X362A/D/Q/S/V.

In some embodiments, the present disclosure provides an engineeredpolypeptide having pNB esterase activity comprises an amino acidsequence having at least 80% identity to a reference sequence of SEQ IDNO: 2 and one or more residue differences as compared to SEQ ID NO: 2selected from: X108L/Y, X193A/D/E/V, X219A/D/L/V, X273A/E/T/V, andX362A/D/Q/S/V.

In some embodiments, the engineered polypeptide having pNB esteraseactivity comprises an amino acid sequence having at least 80% identityto a reference sequence of SEQ ID NO: 2 and a residue difference ascompared to SEQ ID NO: 2 at position X193 selected from: X193A/D/E/V. Insome embodiments, the amino acid residue difference as compared to SEQID NO: 2 at position X193 is X193V.

In some embodiments, the engineered polypeptide having pNB esteraseactivity of the present disclosure comprises an amino acid sequencehaving at least 80% identity to a reference sequence of SEQ ID NO: 2,and an amino acid difference as compared to SEQ ID NO: 2 of X193V, andfurther comprises residue differences as compared to SEQ ID NO: 2 atpositions X219 and X273 selected from X219L/V and X273A/V. In someembodiments, the amino acid sequence further comprises residuedifferences as compared to SEQ ID NO: 2 at positions X108 and X362selected from X108L/Y and X362A/D/Q/S/V. In still further embodiments,the engineered polypeptide further comprises a residue difference ascompared to SEQ ID NO: 2 at position X115 selected from X115Q/W.

In some embodiments, the engineered polypeptide having pNB esteraseactivity of the present disclosure comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, and one ormore residue differences as compared to SEQ ID NO:2 at residue positionsX108, X115, X116, X130, X193, X214, X219, X273, X276, X321, and X362. Insome embodiments, the specific residue differences as compared to SEQ IDNO:2 at residue positions X108, X115, X116, X130, X193, X214, X219,X273, X276, X321, and X362 are selected from: X108L/Y, X115Q/W, X116S,X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L,X321A, and X362A/D/Q/S/V. In some embodiments, the reference sequence isselected from SEQ ID NO: 4, 12, 20, 36, 38, 54, 76, 80, 88, 112, and116. In some embodiments, the reference sequence is SEQ ID NO:4. In someembodiments, the reference sequence is SEQ ID NO:12. In someembodiments, the reference sequence is SEQ ID NO:36. In someembodiments, the reference sequence is SEQ ID NO:38. In someembodiments, the reference sequence is SEQ ID NO:54. In someembodiments, the reference sequence is SEQ ID NO:76. In someembodiments, the reference sequence is SEQ ID NO:80. In someembodiments, the reference sequence is SEQ ID NO:88. In someembodiments, the reference sequence is SEQ ID NO:112. In someembodiments, the reference sequence is SEQ ID NO:116.

In some embodiments, the engineered polypeptide having pNB esteraseactivity of the present disclosure comprises an amino acid sequencehaving at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more identity to a reference sequenceselected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, and acombination of residue differences as compared to SEQ ID NO: 2 selectedfrom: (a) X193V, X219V, and X273A; (b) X108Y, X193D, X219V, X273A, andX362S; (c) X108Y, X193V, X219V, X273A, and X362Q; (d) X108Y, X115Q,X193V, X219L, X273A, and X362Q; and (e) X108Y, X115Q, X193V, X219V,X273A, and X362Q.

In some embodiments, the engineered polypeptides having pNB esteraseactivity disclosed above (and elsewhere herein) can have additionalresidue differences at other residue positions. In some embodiments, theengineered pNB esterases can have 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40,1-45, or 1-50 additional residue differences as compared to SEQ ID NO:2.In some embodiments, the engineered pNB esterases can have 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 30, 35, 40, 45, or 50 additional residue differences. Insome embodiments, the amino acid sequence has additionally 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25residue differences as compared to SEQ ID NO: 2.

In some embodiments, the engineered polypeptide having pNB esteraseactivity of the present disclosure comprises an amino acid sequencehaving at least 80% identity to a reference sequence of SEQ ID NO: 2,one or more amino acid residue difference as compared to SEQ ID NO: 2selected from X108L/Y, X115Q/W, X193A/D/E/V, X219A/D/L/V, X273A/E/T/V,and X362A/D/Q/S/V, and the amino acid sequence further comprises one ormore residue differences as compared to SEQ ID NO: 2 selected from:X116S, X130T, X164T, X214G, X276A/T/L, and X321A. In some embodiments,the amino acid sequence can further comprise a residue difference ascompared to SEQ ID NO: 2 selected from: X49G, X94G, X96S, X227T, X251V,X267R, X271L, X274L, X313F, X322C/Y, X343V, X356R, X359A, X398L, X412E,X437T, X464A, and X481R.

The engineered pNB esterase polypeptide of SEQ ID NO:2 comprises thefollowing seven amino acid differences as compared to the wild-type pNBesterase of Bacillus subtilis (GenBank Access. No.: AAA81915.1,GI:468046): I60V, L144M, P317S, H322R, L334S, M358V, and Y370F.Accordingly, in some embodiments, the present disclosure provides anengineered polypeptide having pNB-esterase activity comprises an aminoacid sequence having at least 80% identity to SEQ ID NO: 2, and of anyof the amino acid differences as compared to SEQ ID NO: 2 as disclosedherein (i.e., as disclosed in the exemplary polypeptides of SEQ ID NO:4-120 of Table 2), but in which the amino acid sequence does notcomprise a residue difference as compared to SEQ ID NO: 2 at a positionselected from: X60, X144, X317, X322, X334, X358, and X370.

As will be appreciated by the skilled artisan, in some embodiments, oneor a combination of residue differences above that is selected can beconserved in the engineered pNB esterases as a core sequence (orfeature), and additional residue differences at other residue positionsincorporated into the core sequence to generate additional engineeredpNB esterase polypeptides with improved properties. Accordingly, it isto be understood for any engineered pNB esterase containing one or asubset of the residue differences above, the present disclosurecontemplates other engineered pNB esterases that comprise the one orsubset of the residue differences, and additionally one or more residuedifferences at the other residue positions disclosed herein. By way ofexample and not limitation, an engineered pNB esterase comprising aresidue difference at residue position X193, can further incorporate oneor more residue differences at the other residue positions, e.g., X108,X115, X116, X130, X214, X219, X273, X276, X321, and X362. Anotherexample is an engineered pNB esterase comprising a residue difference atresidue position X273, which can further comprise one or more residuedifferences at the other residue positions, e.g., X108, X115, X116,X130, X193, X214, X219, X276, X321, and X362. For each of the foregoingembodiments, the engineered pNB esterase can further comprise additionalresidue differences selected from: X108L/Y, X115Q/W, X116S, X130T,X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L, X321A,and X362A/D/Q/S/V.

In some embodiments, the engineered pNB esterase polypeptide is capableof converting the substrate compound (2) to the product compound (1)with at least 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 10fold, or more activity relative to the activity of the referencepolypeptide of SEQ ID NO: 4. In some embodiments, the engineered pNBesterase polypeptide capable of converting the substrate compound (2) tothe product compound (1) with at least 1.2 fold, 1.5 fold, 2 fold, 3fold, 4 fold, 5 fold, 10 fold, or more activity relative to the activityof the reference polypeptide of SEQ ID NO:4 comprises an amino acidsequence having one or more residue differences as compared to SEQ IDNO:4 at a position selected from: X108, X115, X116, X130, X193, X214,X219, X273, X276, X321, and X362, wherein the specific residuedifference is selected from: X108L/Y, X115Q/W, X116S, X130T, X164T,X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L, X321A, andX362A/D/Q/S/V. In some embodiments, the suitable reaction conditions areReaction Conditions A as disclosed in Table 2. In some embodiments, theengineered pNB esterase polypeptide capable of converting the substratecompound (2) to the product compound (1) with at least 1.2 fold theactivity relative to SEQ ID NO:4 comprises an amino acid sequenceselected from: SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 78, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, and 120.

In some embodiments, the engineered pNB esterase having pNB esteraseactivity comprises an amino acid sequence having at least 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, and theamino acid residue differences as compared to SEQ ID NO:2 present in anyone of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, as provided inTable 2.

In addition to the residue positions specified above, any of theengineered pNB esterase polypeptides disclosed herein can furthercomprise other residue differences relative to SEQ ID NO:2 at otherresidue positions, i.e., residue positions other than X108, X115, X116,X130, X193, X214, X219, X273, X276, X321, and X362. Residue differencesat these other residue positions provide for additional variations inthe amino acid sequence without adversely affecting the ability of thepolypeptide to carry out the pNB esterase reaction, such as theconversion of compound (2) to compound (1). Accordingly, in someembodiments, in addition to the amino acid residue differences of anyone of the engineered pNB esterase polypeptides selected from SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, and 120, the sequence can further comprise 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residuedifferences at other amino acid residue positions as compared to the SEQID NO: 2. In some embodiments, the number of amino acid residuedifferences as compared to the reference sequence can be 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 30, 30, 35, 40, 45 or 50 residue positions. The residue differenceat these other positions can include conservative changes ornon-conservative changes. In some embodiments, the residue differencescan comprise conservative substitutions and non-conservativesubstitutions as compared to the wild-type pNB esterase polypeptide ofB. subtilis or the engineered pNB esterase polypeptide of SEQ ID NO: 2.

Amino acid residue differences at other positions relative to thewild-type pNB esterase of B. subtilis or the engineered polypeptide ofSEQ ID NO: 2 and the effect of these differences on enzyme function aredescribed for other engineered pNB esterase polypeptides in U.S. Pat.Nos. 5,906,930 and 5,945,325, each of which is incorporated by referenceherein, and in the following publications, each of which is incorporatedby reference herein: Moore et al., “Directed evolution of apara-nitrobenzyl esterase for aqueous-organic solvents,” NatureBiotechnology 14: 458-467 (1996); Moore et al. “Strategies for the invitro Evolution of Protein Function: Enzyme Evolution by RandomRecombination of Improved Sequences,” J. Mol. Biol. 272:336-347 (1997);Giver et al., “Directed evolution of a thermostable esterase,” Proc.Natl. Acad. Sci. USA 95: 12809-12813 (October 1998). Accordingly, insome embodiments, one or more of the amino acid differences as comparedto the sequence of SEQ ID NO: 2 can also be introduced into anengineered pNB esterase polypeptide of the present disclosure at residuepositions selected from X49, X94, X96, X227, X251, X267, X271, X274,X313, X322, X343, X356, X359, X398, X412, X437, X464, and X481. Inparticular, the amino acid residues at the foregoing positions can beselected from the following: X49G, X94G, X96S, X227T, X251V, X267R,X271L, X274L, X313F, X322C/Y, X343V, X356R, X359A, X398L, X412E, X437T,X464A, and X481R. Guidance on the choice of the amino acid residues atthese residue positions and their effect on desirable enzyme propertiescan be found in the cited references.

In some embodiments, the present disclosure also provides engineered pNBesterase polypeptides that comprise a fragment of any of the engineeredpolypeptides described herein that retains the functional activityand/or improved property of that engineered pNB esterase. Accordingly,in some embodiments, the present disclosure provides a polypeptidefragment having pNB esterase activity, such as in converting compound(2) to compound (1) under suitable reaction conditions, wherein thefragment comprises at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99%of a full-length amino acid sequence of an engineered pNB esterasepolypeptide of the present disclosure, such as an exemplary engineeredpNB esterase polypeptide selected from SEQ ID NO: 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, and 120.

In some embodiments, the engineered pNB esterase polypeptide can have anamino acid sequence comprising a deletion of any one of the engineeredpNB esterase polypeptides described herein, such as the exemplaryengineered polypeptides of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120.Thus, for each and every embodiment of the engineered pNB esterasepolypeptides of the disclosure, the amino acid sequence can comprisedeletions of one or more amino acids, 2 or more amino acids, 3 or moreamino acids, 4 or more amino acids, 5 or more amino acids, 6 or moreamino acids, 8 or more amino acids, 10 or more amino acids, 15 or moreamino acids, or 20 or more amino acids, up to 10% of the total number ofamino acids, up to 10% of the total number of amino acids, up to 20% ofthe total number of amino acids, or up to 30% of the total number ofamino acids of the pNB esterase polypeptides, where the associatedfunctional activity and/or improved properties of the engineered pNBesterase described herein is maintained. In some embodiments, thedeletions can comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10,1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or1-50 amino acid residues. In some embodiments, the number of deletionscan be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acidresidues. In some embodiments, the deletions can comprise deletions of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22,23, 24, or 25 amino acid residues.

In some embodiments, the engineered pNB esterase polypeptide herein canhave an amino acid sequence comprising an insertion as compared to anyone of the engineered pNB esterase polypeptides described herein, suchas the exemplary engineered polypeptides of SEQ ID NO: 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, and 120. Thus, for each and every embodiment of the pNB esterasepolypeptides of the disclosure, the insertions can comprise one or moreamino acids, 2 or more amino acids, 3 or more amino acids, 4 or moreamino acids, 5 or more amino acids, 6 or more amino acids, 8 or moreamino acids, 10 or more amino acids, 15 or more amino acids, 20 or moreamino acids, 30 or more amino acids, 40 or more amino acids, or 50 ormore amino acids, where the associated functional activity and/orimproved properties of the engineered pNB esterase described herein ismaintained. The insertions can be to amino or carboxy terminus, orinternal portions of the pNB esterase polypeptide.

In some embodiments, the engineered pNB esterase polypeptide herein canhave an amino acid sequence comprising a sequence selected from SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, and 120, and optionally one or several (e.g.,up to 3, 4, 5, or up to 10) amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35,40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

In some embodiments, the present disclosure provides an engineeredpolypeptide having pNB esterase activity, which polypeptide comprises anamino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a sequenceselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120, with theproviso that the amino acid sequence is not identical to (that is, itexcludes) any of the exemplary engineered pNB esterase polypeptide aminoacid sequences disclosed in the following publications: U.S. Pat. No.5,906,930; U.S. Pat. No. 5,945,325; Moore et al., “Directed evolution ofa para-nitrobenzyl esterase for aqueous-organic solvents,” NatureBiotechnology 14: 458-467 (1996); Moore et al. “Strategies for the invitro Evolution of Protein Function: Enzyme Evolution by RandomRecombination of Improved Sequences,” J. Mol. Biol. 272:336-347 (1997);and Giver et al., “Directed evolution of a thermostable esterase,” Proc.Natl. Acad. Sci. USA 95: 12809-12813 (October 1998).

In the above embodiments, the suitable reaction conditions for theengineered polypeptides can be those described in Table 2, the Examples,and elsewhere herein.

In some embodiments, the engineered polypeptides of the disclosure canbe in the form of fusion polypeptides in which the engineeredpolypeptides are fused to other polypeptides, such as, by way of exampleand not limitation, antibody tags (e.g., myc epitope), purificationsequences (e.g., His tags for binding to metals), and cell localizationsignals (e.g., secretion signals). Thus, the engineered polypeptidesdescribed herein can be used with or without fusions to otherpolypeptides.

It is to be understood that the engineered polypeptides described hereinare not restricted to the genetically encoded amino acids. In additionto the genetically encoded amino acids, the polypeptides describedherein may be comprised, either in whole or in part, ofnaturally-occurring and/or synthetic non-encoded amino acids. Certaincommonly encountered non-encoded amino acids of which the polypeptidesdescribed herein may be comprised include, but are not limited to: theD-stereoisomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutanic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the engineeredpolypeptides described herein. Non-limiting examples of such protectedamino acids, which in this case belong to the aromatic category, include(protecting groups listed in parentheses), but are not limited to:Arg(tos), Cys(methylbenzyl), Cys (nitropyridinesulfenyl),Glu(δ-benzylester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom),His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe engineered polypeptides described herein may be composed include,but are not limited to, N-methyl amino acids (L-configuration);1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid;azetidine-3-carboxylic acid; homoproline (hPro); and1-aminocyclopentane-3-carboxylic acid.

In some embodiments, the engineered pNB esterase polypeptides can beprovided on a solid support, such as a membrane, resin, solid carrier,or other solid phase material. A solid support can be composed oforganic polymers such as polystyrene, polyethylene, polypropylene,polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well asco-polymers and grafts thereof. A solid support can also be inorganic,such as glass, silica, controlled pore glass (CPG), reverse phase silicaor metal, such as gold or platinum. The configuration of a solid supportcan be in the form of beads, spheres, particles, granules, a gel, amembrane or a surface. Surfaces can be planar, substantially planar, ornon-planar. Solid supports can be porous or non-porous, and can haveswelling or non-swelling characteristics. A solid support can beconfigured in the form of a well, depression, or other container,vessel, feature, or location.

In some embodiments, the engineered polypeptides having pNB esteraseactivity of the present disclosure can be immobilized on a solid supportsuch that they retain their improved activity, and/or other improvedproperties relative to the reference polypeptide of SEQ ID NO: 4. Insuch embodiments, the immobilized engineered pNB esterase polypeptidescan facilitate the biocatalytic conversion of pNB-protected substrate ofcompound (2), or other structurally analogous pNB-protected substratecompounds, to the deprotected product compound (1), imipenem, or acorresponding structural analog product carbapenem, and after thereaction is complete are easily retained (e.g., by retaining beads onwhich polypeptide is immobilized) and then reused or recycled insubsequent reactions. Such immobilized enzyme processes allow forfurther efficiency and cost reduction. Accordingly, it is furthercontemplated that any of the methods of using the engineered pNBesterase polypeptides of the present disclosure can be carried out usingthe same engineered pNB esterase polypeptides bound or immobilized on asolid support.

Methods of enzyme immobilization are well-known in the art. Theengineered pNB esterase polypeptide can be bound non-covalently orcovalently. Various general methods for conjugation and immobilizationof enzymes to solid supports (e.g., resins, membranes, beads, glass,etc.) are well known in the art and described in e.g.: Yi et al.,“Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 onchitosan beads,” Process Biochemistry 42(5): 895-898 (May 2007); Martinet al., “Characterization of free and immobilized (S)-aminotransferasefor acetophenone production,” Applied Microbiology and Biotechnology76(4): 843-851 (September 2007); Koszelewski et al., “Immobilization ofω-transaminases by encapsulation in a sol-gel/celite matrix,” Journal ofMolecular Catalysis B: Enzymatic, 63: 39-44 (April 2010); Truppo et al.,“Development of an Improved Immobilized CAL-B for the EnzymaticResolution of a Key Intermediate to Odanacatib,” Organic ProcessResearch & Development, published online: dx.doi.org/10.1021/op200157c;Hermanson, G. T., Bioconjugate Techniques, Second Edition, AcademicPress (2008); Mateo et al., “Epoxy sepabeads: a novel epoxy support forstabilization of industrial enzymes via very intense multipoint covalentattachment,” Biotechnology Progress 18(3):629-34 (2002); andBioconjugation Protocols: Strategies and Methods, In Methods inMolecular Biology, C. M. Niemeyer ed., Humana Press (2004); thedisclosures of each which are incorporated by reference herein.

Solid supports useful for immobilizing the engineered pNB esterases ofthe present disclosure include but are not limited to beads or resinscomprising polymethacrylate with epoxide functional groups,polymethacrylate with amino epoxide functional groups, styrene/DVBcopolymer or polymethacrylate with octadecyl functional groups.Exemplary solid supports useful for immobilizing the engineered pNBesterases of the present disclosure include, but are not limited to,chitosan beads, Eupergit C, and SEPABEADs (Mitsubishi), including thefollowing different types of SEPABEAD: EC-EP, EC-HFA/S, EXA252, EXE119and EXE120.

In some embodiments, the engineered polypeptides can be in variousforms, for example, such as an isolated preparation, as a substantiallypurified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Theenzymes can be lyophilized, spray-dried, precipitated or be in the formof a crude paste, as further discussed below.

In some embodiments, the engineered polypeptide described herein can beprovided in the form of kits. The enzymes in the kits may be presentindividually or as a plurality of enzymes. The kits can further includereagents for carrying out the enzymatic reactions, substrates forassessing the activity of enzymes, as well as reagents for detecting theproducts. The kits can also include reagent dispensers and instructionsfor use of the kits.

In some embodiments, the engineered polypeptides can be provided on thesolid support in the form of an array in which the polypeptides arearranged in positionally distinct locations. The array can be used totest a variety of substrate compounds for conversion by thepolypeptides. A plurality of supports can be configured on an array atvarious locations, addressable for robotic delivery of reagents, or bydetection methods and/or instruments. Various methods for conjugation tosubstrates, e.g., membranes, beads, glass, etc. are described in, amongothers, Hermanson, G. T., Bioconjugate Techniques, 2^(nd) Edition,Academic Press; (2008), and Bioconjugation Protocols: Strategies andMethods, In Methods in Molecular Biology, C. M. Niemeyer ed., HumanaPress (2004); the disclosures of which are incorporated herein byreference. In some embodiments, the kits of the present disclosureinclude arrays comprising a plurality of different engineeredpolypeptides disclosed herein at different addressable position, whereinthe different polypeptides are different variants of a referencesequence each having at least one different improved enzyme property.Such arrays comprising a plurality of engineered polypeptides andmethods of their use are described in e.g., WO2009008908.

5.4 Polynucleotides Encoding Engineered Polypeptides, Expression Vectorsand Host Cells

In another aspect, the present disclosure provides polynucleotidesencoding the engineered pNB esterase polypeptides described herein. Thepolynucleotides may be operatively linked to one or more heterologousregulatory sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide.Expression constructs containing a heterologous polynucleotide encodingthe engineered pNB esterase can be introduced into appropriate hostcells to express the corresponding pNB esterase polypeptide.

As will be apparent to the skilled artisan, availability of a proteinsequence and the knowledge of the codons corresponding to the variousamino acids provide a description of all the polynucleotides capable ofencoding the subject polypeptides. The degeneracy of the genetic code,where the same amino acids are encoded by alternative or synonymouscodons, allows an extremely large number of nucleic acids to be made,all of which encode the improved pNB esterase enzymes. Thus, havingknowledge of a particular amino acid sequence, those skilled in the artcould make any number of different nucleic acids by simply modifying thesequence of one or more codons in a way which does not change the aminoacid sequence of the protein. In this regard, the present disclosurespecifically contemplates each and every possible variation ofpolynucleotides that could be made encoding the polypeptides describedherein by selecting combinations based on the possible codon choices,and all such variations are to be considered specifically disclosed forany polypeptide described herein, including the amino acid sequencespresented in Table 2, and disclosed in the Sequence Listing incorporatedby reference herein as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and 120.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used for expression in bacteria; preferredcodons used in yeast are used for expression in yeast; and preferredcodons used in mammals are used for expression in mammalian cells. Insome embodiments, all codons need not be replaced to optimize the codonusage of the pNB esterases since the natural sequence will comprisepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the pNB esterase enzymes may contain preferredcodons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codonpositions of the full length coding region.

In some embodiments, as described above, the polynucleotide encodes anengineered polypeptide having pNB esterase activity with the propertiesdisclosed herein, such as the ability to convert the substrate compound(2) to the product compound (1), where the polypeptide comprises anamino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to areference sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, and120, and one or more residue differences as compared to the referencepolypeptide of SEQ ID NO:2 at residue positions X108, X115, X116, X130,X193, X214, X219, X273, X276, X321, and X362. In some embodiments, thespecific residue differences as compared to SEQ ID NO:2 at residuepositions X108, X115, X116, X130, X193, X214, X219, X273, X276, X321,and X362 are selected from: X108L/Y, X115Q/W, X116S, X130T, X164T,X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L, X321A, andX362A/D/Q/S/V. In some embodiments, the reference sequence is selectedfrom SEQ ID NO: 4, 12, 20, 36, 38, 54, 76, 80, 88, 112, and 116. In someembodiments, the reference sequence is SEQ ID NO:4. In some embodiments,the reference sequence is SEQ ID NO:12. In some embodiments, thereference sequence is SEQ ID NO:36. In some embodiments, the referencesequence is SEQ ID NO:38. In some embodiments, the reference sequence isSEQ ID NO:54. In some embodiments, the reference sequence is SEQ IDNO:76. In some embodiments, the reference sequence is SEQ ID NO:80. Insome embodiments, the reference sequence is SEQ ID NO:88. In someembodiments, the reference sequence is SEQ ID NO:112. In someembodiments, the reference sequence is SEQ ID NO:116.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having pNB esterase activity with the properties disclosedherein, wherein the polypeptide comprises an amino acid sequence havingat least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to reference sequence SEQID NO:2 and one or more residue differences as compared to SEQ ID NO: 2at residue positions selected from as compared to SEQ ID NO:2 at residuepositions X108, X115, X193, X219, X273, X276, and X362 are selectedfrom: X108L/Y, X115Q/W, X193A/D/E/V, X219A/D/L/V, X273A/E/T/V,X276A/T/L, and X362A/D/Q/S/V.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having pNB esterase activity, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to reference sequence SEQ ID NO:2 and at least acombination of residue differences as compared to SEQ ID NO: 2 selectedfrom: (a) X193V, X219V, and X273A; (b) X108Y, X193D, X219V, X273A, andX362S; (c) X108Y, X193V, X219V, X273A, and X362Q; (d) X108Y, X115Q,X193V, X219L, X273A, and X362Q; and (e) X108Y, X115Q, X193V, X219V,X273A, and X362Q.

In some embodiments, the polynucleotide encodes an engineeredpolypeptide having pNB esterase activity, wherein the polypeptidecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identityto a reference polypeptide selected from any one of SEQ ID NO: 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, and 120, with the proviso that the amino acid sequencecomprises any one of the set of residue differences as compared to SEQID NO: 2 contained in any one of the polypeptide sequences of SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, and 120, as listed in Table 2.

In some embodiments, the polynucleotide encoding the engineered pNBesterase comprises a polynucleotide sequence selected from SEQ ID NO: 3,5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, and 119.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a reference polynucleotide sequenceselected from SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, and 119, or acomplement thereof, and encodes a polypeptide having pNB esteraseactivity with one or more of the improved properties described herein.In some embodiments, the polynucleotide capable of hybridizing underhighly stringent conditions encodes a pNB esterase polypeptidecomprising an amino acid sequence that has one or more residuedifferences as compared to SEQ ID NO: 2 at residue positions selectedfrom X108, X115, X116, X130, X193, X214, X219, X273, X276, X321, andX362, and optionally wherein the specific residue differences ascompared to SEQ ID NO:2 are selected from: X108L/Y, X115Q/W, X116S,X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L,X321A, and X362A/D/Q/S/V.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity atthe nucleotide level to a reference polynucleotide encoding theengineered pNB esterase. In some embodiments, the referencepolynucleotide sequence is selected from SEQ ID NO: 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,and 119.

An isolated polynucleotide encoding any of the engineered pNB esterasepolypeptides herein may be manipulated in a variety of ways to providefor expression of the polypeptide. In some embodiments, thepolynucleotides encoding the polypeptides can be provided as expressionvectors where one or more control sequences is present to regulate theexpression of the polynucleotides and/or polypeptides. Manipulation ofthe isolated polynucleotide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art. Guidance isprovided in e.g., Sambrook et al., 2001, “Molecular Cloning: ALaboratory Manual,” 3^(rd) Ed., Cold Spring Harbor Laboratory Press; andCurrent Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub.Associates, 1998, and updates to 2006.

In some embodiments, the control sequences include among others,promoter, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator.Suitable promoters can be selected based on the host cells used. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, Streptomyces coelicoloragarase gene (dagA), Bacillus subtilis levansucrase gene (sacB),Bacillus licheniformis alpha-amylase gene (amyL), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillusamyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformispenicillinase gene (penP), Bacillus subtilis xylA and xylB genes, andprokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. NatlAcad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer etal., 1983, Proc. Natl Acad. Sci. USA 80: 21-25). Exemplary promoters forfilamentous fungal host cells, include promoters obtained from the genesfor Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-likeprotease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), and mutant, truncated,and hybrid promoters thereof. Exemplary yeast cell promoters can be fromthe genes can be from the genes for Saccharomyces cerevisiae enolase(ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomycescerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphatedehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention. For example, exemplary transcription terminatorsfor filamentous fungal host cells can be obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. Any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used for expression of the engineeredpolypeptides. Effective signal peptide coding regions for bacterial hostcells are the signal peptide coding regions obtained from the genes forBacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57:109-137.Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase. Usefulsignal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is referred to as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila lactase (WO 95/33836). Whereboth signal peptide and propeptide regions are present at the aminoterminus of a polypeptide, the propeptide region is positioned next tothe amino terminus of a polypeptide and the signal peptide region ispositioned next to the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

In another aspect, the present disclosure is also directed to arecombinant expression vector comprising a polynucleotide encoding anengineered pNB esterase polypeptide, and one or more expressionregulating regions such as a promoter and a terminator, a replicationorigin, etc., depending on the type of hosts into which they are to beintroduced. The various nucleic acid and control sequences describedabove may be joined together to produce a recombinant expression vectorwhich may include one or more convenient restriction sites to allow forinsertion or substitution of the nucleic acid sequence encoding thepolypeptide at such sites. Alternatively, the nucleic acid sequence ofthe present disclosure may be expressed by inserting the nucleic acidsequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector preferably contains one or more selectablemarkers, which permit easy selection of transformed cells. A selectablemarker is a gene the product of which provides for biocide or viralresistance, resistance to heavy metals, prototrophy to auxotrophs, andthe like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers, whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol (Example 1) or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferases), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an engineered pNB esterasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe pNB esterase enzyme in the host cell. Host cells for use inexpressing the polypeptides encoded by the expression vectors of thepresent invention are well known in the art and include but are notlimited to, bacterial cells, such as E. coli, Vibrio fluvialis,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. An exemplary host cells are Escherichiacoli W3110 (ΔfhuA) and BL21.

Accordingly, in another aspect, the present disclosure provides methodsof manufacturing the engineered pNB esterase polypeptides, where themethod can comprise culturing a host cell capable of expressing apolynucleotide encoding the engineered pNB esterase polypeptide underconditions suitable for expression of the polypeptide. The method canfurther comprise isolated or purifying the expressed pNB esterasespolypeptide, as described herein.

Appropriate culture mediums and growth conditions for theabove-described host cells are well known in the art. Polynucleotidesfor expression of the pNB esterase may be introduced into cells byvarious methods known in the art. Techniques include, among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.

For the embodiments herein, the engineered polypeptides andcorresponding polynucleotides can be obtained using methods used bythose skilled in the art. The parental polynucleotide sequence encodingthe wild-type pNB esterase polypeptide of Bacillus subtilis is disclosedin Zock et al., “The Bacillus subtilis pnbA gene encoding p-nitrobenzylesterase: cloning, sequence and high-level expression in Escherichiacoli,” Gene 151: 37-43 (1994), and U.S. Pat. No. 5,468,632, and methodsof generating engineered pNB esterase polypeptides with improvedstability are disclosed in U.S. Pat. Nos. 5,906,930 and 5,945,325, eachof which is incorporated by reference herein, and in the followingpublications, each of which is incorporated by reference herein: Mooreet al., “Directed evolution of a para-nitrobenzyl esterase foraqueous-organic solvents,” Nature Biotechnology 14: 458-467 (1996);Moore et al. “Strategies for the in vitro Evolution of Protein Function:Enzyme Evolution by Random Recombination of Improved Sequences,” J. Mol.Biol. 272:336-347 (1997); Giver et al., “Directed evolution of athermostable esterase,” Proc. Natl. Acad. Sci. USA 95: 12809-12813(October 1998).

The engineered pNB esterases with the properties disclosed herein can beobtained by subjecting the polynucleotide encoding the naturallyoccurring or engineered pNB esterase to mutagenesis and/or directedevolution methods known in the art, and as described herein. Anexemplary directed evolution technique is mutagenesis and/or DNAshuffling as described in Stemmer, 1994, Proc. Natl. Acad. Sci. USA91:10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO00/42651; WO 01/75767 and U.S. Pat. No. 6,537,746. Other directedevolution procedures that can be used include, among others, staggeredextension process (StEP), in vitro recombination (Zhao et al., 1998,Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell et al., 1994, PCRMethods Appl. 3:S136-S140), and cassette mutagenesis (Black et al.,1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesis and directedevolution techniques useful for the purposes herein are also describedin the following references: Ling, et al., 1997, Anal. Biochem.254(2):157-78; Dale et al., 1996, “Oligonucleotide-directed randommutagenesis using the phosphorothioate method,” In Methods Mol. Biol.57:369-74; Smith, 1985, Ann. Rev. Genet. 19:423-462; Botstein et al.,1985, Science 229:1193-1201; Carter, 1986, Biochem. J. 237:1-7; Krameret al., 1984, Cell, 38:879-887; Wells et al., 1985, Gene 34:315-323;Minshull et al., 1999, Curr Opin Chem Biol 3:284-290; Christians et al.,1999, Nature Biotech 17:259-264; Crameri et al., 1998, Nature391:288-291; Crameri et al., 1997, Nature Biotech 15:436-438; Zhang etal., 1997, Proc Natl Acad Sci USA 94:45-4-4509; Crameri et al., 1996,Nature Biotech 14:315-319; Stemmer, 1994, Nature 370:389-391; Stemmer,1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO 97/0078; WO97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S. Pat. No.6,537,746. All publications are incorporated herein by reference.

The clones obtained following mutagenesis treatment can be screened forengineered pNB esterases having a desired improved enzyme property. Forexample, where the improved enzyme property desired is thermostability,enzyme activity may be measured after subjecting the enzyme preparationsto a defined temperature and measuring the amount of enzyme activityremaining after heat treatments. Clones containing a polynucleotideencoding a pNB esterase are then isolated, sequenced to identify thenucleotide sequence changes (if any), and used to express the enzyme ina host cell. Measuring enzyme activity from the expression libraries canbe performed using the standard biochemistry techniques, such as HPLCanalysis.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides disclosedherein can be prepared by chemical synthesis using, e.g., the classicalphosphoramidite method described by Beaucage et al., 1981, Tet Lett22:1859-69, or the method described by Matthes et al., 1984, EMBO J.3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors.

Accordingly, in some embodiments, a method for preparing the engineeredpNB esterase polypeptide can comprise: (a) synthesizing a polynucleotideencoding a polypeptide comprising an amino acid sequence selected fromSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and 120 and having one or moreresidue differences as compared to SEQ ID NO: 2 at residue positionsselected from: X108, X115, X116, X130, X193, X214, X219, X273, X276,X321, and X362, and wherein the specific residue differences as comparedto SEQ ID NO:2 optionally are selected from: X108L/Y, X115Q/W, X116S,X130T, X164T, X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L,X321A, and X362A/D/Q/S/V; and (b) expressing the pNB esterasepolypeptide encoded by the polynucleotide.

In some embodiments of the method, the amino acid sequence encoded bythe polynucleotide can optionally have one or several (e.g., up to 3, 4,5, or up to 10) amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acidresidue deletions, insertions and/or substitutions. In some embodiments,the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35,40, 45, or 50 amino acid residue deletions, insertions and/orsubstitutions. In some embodiments, the amino acid sequence hasoptionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18,20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertionsand/or substitutions. In some embodiments, the substitutions can beconservative or non-conservative substitutions.

The expressed engineered pNB esterase can be measured for the desiredimproved property, e.g., activity, selectivity, stability, and/orproduct tolerance, in the conversion of compound (2) to compound (1) byany of the assay conditions described herein.

In some embodiments, any of the engineered pNB esterase enzymesexpressed in a host cell can be recovered from the cells and or theculture medium using any one or more of the well known techniques forprotein purification, including, among others, lysozyme treatment,sonication, filtration, salting-out, ultra-centrifugation, andchromatography. Suitable solutions for lysing and the high efficiencyextraction of proteins from bacteria, such as E. coli, are provided inTable 2 and the Examples, and also commercially available, e.g.,CelLytic B™ from Sigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the pNB esterase polypeptideinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,and affinity chromatography. Conditions for purifying a particularenzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art.

In some embodiments, affinity techniques may be used to isolate theimproved pNB esterase enzymes. For affinity chromatography purification,any antibody which specifically binds the pNB esterase polypeptide maybe used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with a pNB esterase polypeptide, or a fragment thereof. ThepNB esterase polypeptide or fragment may be attached to a suitablecarrier, such as BSA, by means of a side chain functional group orlinkers attached to a side chain functional group.

5.7 Methods of Using the Engineered pNB Esterase Polypeptides

As noted above, the engineered pNB esterase polypeptides of the presentdisclosure were evolved to efficiently convert the pNB-protectedsubstrate of compound (2) to the corresponding product compound (1),imipenem, under suitable reaction conditions. The structural features ofthe engineered pNB esterase polypeptides allow for the conversion of thepNB-protected substrate of compound (2) to their correspondingdeprotected product of compound (1), imipenem. Accordingly, in anotheraspect the present disclosure provides a process for preparingcarbapenem antibiotic of compound (1), imipenem, or a salt or hydrate ofcompound (1),

wherein the method comprises contacting a substrate compound (2), or asalt or hydrate of compound (2),

with an engineered pNB esterase polypeptide of the present disclosureunder suitable reaction conditions.

The structural features of the engineered pNB esterase polypeptides canalso provide engineered pNB esterases capable of converting of otherpNB-protected carbapenem substrates that are structural analogs ofcompound (2). Accordingly, in another aspect, the present disclosureprovides processes using the engineered pNB esterase polypeptides tocarry out a deprotection reaction in which a pNB group is removed from apNB-protected carbapenem compound. Generally, the process for performingthe biocatalytic pNB deprotection reaction comprises contacting orincubating an engineered pNB esterase polypeptide of the disclosure withthe pNB-protected compound with under reaction conditions suitable fordeprotecting the carbapenem precursor and yielding the desiredcarbapenem compound.

For the foregoing processes, any of the engineered pNB esterasepolypeptides described herein can be used. By way of example and withoutlimitation, in some embodiments, the process can use an engineeredpolypeptide having pNB esterase activity of the present disclosurecomprises an amino acid sequence having at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentity to a reference sequence selected from SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, and 120, and one or more residue differences as compared toSEQ ID NO:2 at residue positions selected from X108, X115, X116, X130,X193, X214, X219, X273, X276, X321, and X362. In some embodiments, thespecific residue differences as compared to SEQ ID NO:2 at residuepositions X108, X115, X116, X130, X193, X214, X219, X273, X276, X321,and X362 are selected from: X108L/Y, X115Q/W, X116S, X130T, X164T,X193A/D/E/V, X214G, X219A/D/L/V, X273A/E/T/V, X276A/T/L, X321A, andX362A/D/Q/S/V. In some embodiments, the reference sequence is selectedfrom SEQ ID NO: 4, 12, 20, 36, 38, 54, 76, 80, 88, 112, and 116. In someembodiments, the reference sequence is SEQ ID NO:4. In some embodiments,the reference sequence is SEQ ID NO:12. In some embodiments, thereference sequence is SEQ ID NO:36. In some embodiments, the referencesequence is SEQ ID NO:38. In some embodiments, the reference sequence isSEQ ID NO:54. In some embodiments, the reference sequence is SEQ IDNO:76. In some embodiments, the reference sequence is SEQ ID NO:80. Insome embodiments, the reference sequence is SEQ ID NO:88. In someembodiments, the reference sequence is SEQ ID NO:112. In someembodiments, the reference sequence is SEQ ID NO:116.

In some embodiments, exemplary pNB esterase polypeptides capable ofcarrying out the processes herein can be a polypeptide comprising anamino acid sequence selected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,and 120. Guidance on the choice and use of the engineered pNB esterasepolypeptides is provided in the descriptions herein, for example Table 2and the Examples.

In the embodiments herein and illustrated in the Examples, variousranges of suitable reaction conditions that can be used, including butnot limited, to ranges of pH, temperature, buffer, solvent system,substrate loading, polypeptide loading, pressure, and reaction time.Further suitable reaction conditions for carrying out the process forbiocatalytic conversion of substrate compounds to product compoundsusing an engineered pNB esterase polypeptide described herein can bereadily optimized in view of the guidance provided herein by routineexperimentation that includes, but is not limited to, contacting theengineered pNB esterase polypeptide and substrate compound underexperimental reaction conditions of concentration, pH, temperature,solvent conditions, and detecting the product compound.

Substrate compound in the reaction mixtures can be varied, taking intoconsideration, for example, the desired amount of product compound, theeffect of substrate concentration on enzyme activity, stability ofenzyme under reaction conditions, and the percent conversion ofsubstrate to product. In some embodiments, the suitable reactionconditions comprise a substrate compound loading of at least about 0.5to about 200 g/L, 1 to about 200 g/L, about 5 to about 150 g/L, about 10to about 100 g/L, about 20 to about 100 g/L, or about 50 to about 100g/L. In some embodiments, the suitable reaction conditions comprise asubstrate compound loading of at least about 0.5 g/L, at least about 1g/L, at least about 5 g/L, at least about 10 g/L, at least about 15 g/L,at least about 20 g/L, at least about 30 g/L, at least about 50 g/L, atleast about 75 g/L, at least about 100 g/L, at least about 150 g/L or atleast about 200 g/L, or even greater. The values for substrate loadingsprovided herein are based on the molecular weight of compound (2),however it also contemplated that the equivalent molar amounts ofvarious hydrates and salts of compound (2) also can be used in theprocess. In addition, structural analogs of the substrate of compound(2), can also be used in appropriate amounts, in light of the amountsused for the substrate of compound (2).

In carrying out the reactions described herein, the engineered pNBesterase polypeptide may be added to the reaction mixture in the form ofa purified enzyme, whole cells transformed with gene(s) encoding theenzyme, and/or as cell extracts and/or lysates of such cells. Wholecells transformed with gene(s) encoding the engineered pNB esteraseenzyme or cell extracts, lysates thereof, and isolated enzymes may beemployed in a variety of different forms, including solid (e.g.,lyophilized, spray-dried, and the like) or semisolid (e.g., a crudepaste). The cell extracts or cell lysates may be partially purified byprecipitation (ammonium sulfate, polyethyleneimine, heat treatment orthe like), followed by a desalting procedure prior to lyophilization(e.g., ultrafiltration, dialysis, and the like). Any of the cellpreparations may be stabilized by crosslinking using known crosslinkingagents, such as, for example, glutaraldehyde, or immobilization to asolid phase (e.g., Eupergit C, and the like).

The gene(s) encoding the engineered pNB esterase polypeptides can betransformed into host cell separately or together into the same hostcell. For example, in some embodiments one set of host cells can betransformed with gene(s) encoding one engineered pNB esterasepolypeptide and another set can be transformed with gene(s) encodinganother engineered pNB esterase polypeptide. Both sets of transformedcells can be utilized together in the reaction mixture in the form ofwhole cells, or in the form of lysates or extracts derived therefrom. Inother embodiments, a host cell can be transformed with gene(s) encodingmultiple engineered pNB esterase polypeptides. In some embodiments theengineered polypeptides can be expressed in the form of secretedpolypeptides and the culture medium containing the secreted polypeptidescan be used for the pNB esterase reaction.

The enhancements in activity and/or product selectivity of theengineered pNB esterase polypeptides disclosed herein provide forprocesses wherein higher percentage conversion can be achieved withlower concentrations of the engineered polypeptide. In some embodimentsof the process, the suitable reaction conditions comprise an engineeredpolypeptide concentration of about 0.01 to about 50 g/L; about 0.05 toabout 50 g/L; about 0.1 to about 40 g/L; about 1 to about 40 g/L; about2 to about 40 g/L; about 5 to about 40 g/L; about 5 to about 30 g/L;about 0.1 to about 10 g/L; about 0.5 to about 10 g/L; about 1 to about10 g/L; about 0.1 to about 5 g/L; about 0.5 to about 5 g/L; or about 0.1to about 2 g/L. In some embodiments, the pNB esterase polypeptide isconcentration at about 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20,25, 30, 35, 40, or 50 g/L.

During the course of the pNB esterase reactions, the pH of the reactionmixture may change. The pH of the reaction mixture may be maintained ata desired pH or within a desired pH range. This may be done by adding anacid or base, before and/or during the course of the reaction.Alternatively, the pH may be controlled by using a buffer. Accordingly,in some embodiments, the reaction condition comprises a buffer. Suitablebuffers to maintain desired pH ranges are known in the art and include,by way of example and not limitation, phosphate,2-(N-morpholino)ethanesulfonic acid (MES), borate, carbonate,triethanolamine (TEA), and the like. In some embodiments, the buffer isborate. In some embodiments of the process, the suitable reactionconditions comprise a buffer solution of MES, where the MESconcentration is from about 0.01 to about 0.4 M, 0.05 to about 0.4 M,0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In some embodiments,the reaction condition comprises a MES concentration of about 0.01,0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.3, or0.4 M. In some embodiments, the reaction conditions comprise water as asuitable solvent with no buffer present.

In the embodiments of the process, the reaction conditions can comprisea suitable pH. The desired pH or desired pH range can be maintained byuse of an acid or base, an appropriate buffer, or a combination ofbuffering and acid or base addition. The pH of the reaction mixture canbe controlled before and/or during the course of the reaction. In someembodiments, the suitable reaction conditions comprise a solution pHfrom about 5 to about 12, pH from about 6 to about 9, pH from about 6 toabout 8, pH from about 6.5 to about 7.5, or pH from about 7 to about 8.In some embodiments, the reaction conditions comprise a solution pH ofabout 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12.

In the embodiments of the processes herein, a suitable temperature canbe used for the reaction conditions, for example, taking intoconsideration the increased reaction rate at higher temperatures, andthe activity of the enzyme during the reaction time period. For example,the engineered polypeptides of the present disclosure have increasedstability relative to naturally occurring pNB esterase polypeptide e.g.,the wild-type polypeptide of SEQ ID NO: 2, which allow the engineeredpolypeptides to be used at higher temperatures for increased conversionrates and improved substrate solubility characteristics. Accordingly, insome embodiments, the suitable reaction conditions comprise atemperature of about 5° C. to about 65° C., about 10° C. to about 60°C., about 15° C. to about 55° C., about 15° C. to about 45° C., about15° C. to about 35° C., about 20° C. to about 55° C., or about 30° C. toabout 60° C. In some embodiments, the suitable reaction conditionscomprise a temperature of about 5° C., about 10° C., about 15° C., about20° C., about 25° C., about 30° C., about 35° C., about 40° C., about45° C., about 50° C., about 55° C., about 60° C., about 65° C., or about70° C.

In some embodiments, higher temperatures (e.g., above 25° C.) can resultin increased undesirable side-products, such as the β-lactam ring-openeddiacid imipenem side-product of compound (3). Accordingly, in someembodiments, the suitable reaction conditions comprise a temperature ofabout 5° C. to about 30° C., about 10° C. to about 25° C., about 10° C.to about 20° C., or about 15° C. to about 20° C. In some embodiments,the suitable reaction conditions comprise a temperature of about 5° C.,about 10° C., about 15° C., about 20° C., about 25° C., or about 30° C.

In some embodiments, the temperature during the enzymatic reaction canbe maintained at a temperature throughout the course of the reaction oradjusted over a temperature profile during the course of the reaction.

The processes herein are generally carried out in a solvent. Suitablesolvents include water, aqueous buffer solutions, organic solvents,polymeric solvents, and/or co-solvent systems, which generally compriseaqueous solvents, organic solvents and/or polymeric solvents. Theaqueous solvent (water or aqueous co-solvent system) may be pH-bufferedor unbuffered. In some embodiments, the processes are generally carriedout in an aqueous co-solvent system comprising an organic solvent (e.g.,ethanol, isopropanol (IPA), dimethylformamide (DMF), dimethyl sulfoxide(DMSO), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methylt-butyl ether (MTBE), toluene, and the like), ionic or polar solvents(e.g., 1 ethyl 4 methylimidazolium tetrafluoroborate, 1 butyl 3methylimidazolium tetrafluoroborate, 1 butyl 3 methylimidazoliumhexafluorophosphate, glycerol, polyethylene glycol, and the like). Ingeneral, the co-solvent component of an aqueous co-solvent system ischosen such that it does not adversely inactivate the pNB esteraseenzyme under the reaction conditions. Appropriate co-solvent systems canbe readily identified by measuring the enzymatic activity of thespecified engineered pNB esterase enzyme with a defined substrate ofinterest in the candidate solvent system, utilizing an enzyme activityassay, such as those described herein. The non-aqueous co-solventcomponent of an aqueous co-solvent system may be miscible with theaqueous component, providing a single liquid phase, or may be partlymiscible or immiscible with the aqueous component, providing two liquidphases. Exemplary aqueous co-solvent systems can comprise water and oneor more co-solvents selected from an organic solvent, polar solvent, andpolyol solvent. In some embodiments, the co-solvent can be a polarsolvent, such as DMF, DMSO, or lower alcohol.

In some embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent, where the co-solvent comprises DMF atabout 1% to about 80% (v/v), about 1 to about 70% (v/v), about 2% toabout 60% (v/v), about 5% to about 40% (v/v), 10% to about 40% (v/v),10% to about 30% (v/v), or about 10% to about 20% (v/v). In someembodiments of the process, the suitable reaction conditions comprise anaqueous co-solvent comprising DMF at least about 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% (v/v). Insome embodiments of the process, the suitable reaction conditionscomprise an aqueous co-solvent comprising DMF of from about 5% (v/v) toabout 45% (v/v), from about 10% (v/v) to about 30% (v/v), and in someembodiments a DMF concentration of about 15% (v/v).

The quantities of reactants used in the pNB esterase reaction willgenerally vary depending on the quantities of product desired, andconcomitantly the amount of pNB esterase substrate employed. Thosehaving ordinary skill in the art will readily understand how to varythese quantities to tailor them to the desired level of productivity andscale of production.

In some embodiments, the order of addition of reactants is not critical.The reactants may be added together at the same time to a solvent (e.g.,monophasic solvent, biphasic aqueous co-solvent system, and the like),or alternatively, some of the reactants may be added separately, andsome together at different time points. For example, the cofactor, pNBesterase, and pNB esterase substrate may be added first to the solvent.

The solid reactants (e.g., enzyme, salts, substrate compounds, etc.) maybe provided to the reaction in a variety of different forms, includingpowder (e.g., lyophilized, spray dried, and the like), solution,emulsion, suspension, and the like. The reactants can be readilylyophilized or spray dried using methods and equipment that are known tothose having ordinary skill in the art. For example, the proteinsolution can be frozen at −80° C. in small aliquots, then added to apre-chilled lyophilization chamber, followed by the application of avacuum.

For improved mixing efficiency when an aqueous co-solvent system isused, the pNB esterase and cofactor may be added and mixed into theaqueous phase first. The organic phase may then be added and mixed in,followed by addition of the pNB esterase substrate. Alternatively, thepNB esterase substrate may be premixed in the organic phase, prior toaddition to the aqueous phase.

The pNB esterase reaction is generally allowed to proceed until furtherconversion of pNB-protected substrate to product does not changesignificantly with reaction time, e.g., less than 10% of substrate beingconverted, or less than 5% of substrate being converted. In someembodiments, the reaction is allowed to proceed until there is completeor near complete conversion of pNB-protected substrate to thedeprotected product compound. Transformation of substrate to product canbe monitored using known methods by detecting substrate and/or product.Suitable methods include gas chromatography, HPLC, and the like.Conversion yields of the deprotected product compound generated in thereaction mixture are generally greater than about 50%, may also begreater than about 60%, may also be greater than about 70%, may also begreater than about 80%, may also be greater than 90%, and may be greaterthan about 97%. In some embodiments, the methods for preparing thedeprotected imipenem compound (1) using an engineered pNB esterasepolypeptide under suitable reaction conditions results in at least about91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater conversion of thepNB-protected substrate of compound (2), to the deprotected imipenemproduct of compound (1) in about 48 h or less, in about 36 h or less, inabout 24 h or less, or even less time.

In some embodiments of the process, the suitable reaction conditionscomprise a substrate loading of at least about 5 g/L, 10 g/L, 20 g/L, 30g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, and wherein theprocess results in at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater conversion of substratecompound to product compound in about 48 h or less, in about 36 h orless, or in about 24 h or less.

In a further embodiment of the processes, the suitable reactionconditions can comprise an initial substrate loading to the reactionsolution which is then contacted by the engineered pNB esterasepolypeptide. The reaction solution is then further supplemented withadditional substrate compound as a continuous addition over time at arate of at least about 1 g/L/h, at least about 2 g/L/h, at least about 4g/L/h, at least about 6 g/L/h, or higher. Thus, according to thesesuitable reaction conditions, polypeptide is added to a solution havingan initial substrate loading of at least about 5 g/L, 10 g/L, 20 g/L, 30g/L, or 40 g/L. This addition of polypeptide is then followed bycontinuous addition of further substrate to the solution at a rate ofabout 2 g/L/h, 4 g/L/h, or 6 g/L/h until a much higher final substrateloading of at least about 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100g/L, 150 g/L, 200 g/L or more, is reached. Accordingly, in someembodiments of the process, the suitable reaction conditions compriseaddition of the polypeptide to a solution having an initial substrateloading of at least about 20 g/L, 30 g/L, or 40 g/L followed by additionof further substrate to the solution at a rate of about 2 g/L/h, 4g/L/h, or 6 g/L/h until a final substrate loading of at least about 30g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached. Thissubstrate supplementation reaction condition allows for higher substrateloadings to be achieved while maintaining high rates of conversion ofthe pNB-protected substrate to the deprotected product of at least about91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater.

In some embodiments of the processes, the pNB esterase reaction cancomprise the following suitable reaction conditions: (a) substrateloading at about 2 g/L to 200 g/L; (b) about 0.1 to 10 g/L of engineeredpNB esterase polypeptide; (c) about 0.05 to 0.5 M MES buffer; (d) about5% to about 20% (v/v) DMF co-solvent; (e) pH of about 6 to 8; and (f)temperature of about 10 to 35° C.

In some embodiments of the processes, the pNB esterase reaction cancomprise the following suitable reaction conditions: (a) substrateloading at about 5 g/L to 100 g/L; (b) about 2 to 5 g/L of engineeredpNB esterase polypeptide; (c) about 0.1 M MES buffer; (d) about 15%(v/v) DMF co-solvent; (e) pH of about 7; and (f) temperature of about15° C.

In some embodiments, additional reaction components or additionaltechniques carried out to supplement the reaction conditions. These caninclude taking measures to stabilize or prevent inactivation of the pNBesterase polypeptide, reduce product compound inhibition, reduceundesirable side-product production, and/or shift reaction equilibriumto product compound formation.

In further embodiments, any of the above described processes for theconversion of substrate compound to product compound can furthercomprise one or more steps selected from: extraction, isolation,purification, and crystallization of product compound. Methods,techniques, and protocols for extracting, isolating, purifying, and/orcrystallizing the product compound from the biocatalytic reactionmixtures produced by the above disclosed methods are known to theordinary artisan and/or accessed through routine experimentation.Additionally, illustrative methods are provided in the Examples below.

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

6. EXAMPLES Example 1: Synthesis, Optimization, and Screening ofEngineered pNB Esterase Polypeptides

Gene Synthesis and Optimization:

The polynucleotide sequence encoding the 489 amino acid wild-type pNBesterase polypeptide from Bacillus subtilis (Genbank Acc. No.AAA81915.1, GI: 468046) was codon optimized for expression in E. colitogether with nucleotide changes encoding the following 7 amino acidsubstitutions: I60V, L144M, P317S, H322R, L334S, M358V, and Y370F. Thiscodon-optimized gene, disclosed herein as SEQ ID NO: 1, was synthesizedand cloned into a pCK110900 vector system (see e.g., US PatentApplication Publication 20060195947, which is hereby incorporated byreference herein) and subsequently expressed in E. coli W3110fhuA. TheE. coli W3110 expresses the pNB esterase polypeptides as anintracellular protein under the control of the lac promoter. The initialengineered polypeptide of SEQ ID NO: 2 did not have detectable activityin the conversion of the pNB-protected imipenem substrate compound (2)to imipenem of compound (1). Based on structural modeling of compound(2) of the active site of the wild-type pNB esterase, the engineeredpolypeptide of SEQ ID NO: 2 was further modified with the amino acidsubstitution M193A, resulting in the engineered pNB esterase polypeptideof SEQ ID NO: 4, which was found to have activity in the conversion ofcompound (2) to compound (1). The polynucleotide of SEQ ID NO: 3 (whichencodes the engineered pNB esterase polypeptide of SEQ ID NO: 4) thenwas used as the starting backbone for further optimization usingstandard methods of directed evolution via iterative variant librarygeneration by gene synthesis followed by screening and sequencing of thehits to generate genes encoding engineered pNB esterases capable ofconverting compound (2) to compound (1) with enhanced enzyme propertiesrelative to the engineered polypeptide of SEQ ID NO: 4. The resultingengineered pNB esterase polypeptide sequences and specific mutations andrelative activities are listed in Table 2 and the Sequence Listing.

Example 2: Production of Engineered pNB Esterases

The engineered pNB esterase polypeptides were produced in host E. coliW3110 as an intracellular protein expressed under the control of the lacpromoter. The polypeptide accumulates primarily as a soluble cytosolicactive enzyme. A shake-flask procedure is used to generate engineeredpolypeptide powders that can be used in activity assays or biocatalyticprocesses disclosed herein.

High-Throughput Growth and Expression.

Cells are picked and grown overnight in LB media containing 1% glucoseand 30 μg/mL chloramphenicol (CAM) under culture conditions of 30° C.,200 rpm, and 85% humidity. A 20 μL aliquot of overnight growth aretransferred to a deep well plate containing 380 μL 2×YT growth mediacontaining 30 μg/mL CAM, 1 mM IPTG, and incubated for ˜18 h at 30° C.,200 rpm, and 85% humidity. Subculture TB media is made up of TB media(380 μL/well), 30 μg/mL CAM, and 1 mM IPTG. Cell cultures arecentrifuged at 4000 rpm, 4° C. for 10 minutes, and the supernatant mediadiscarded. Cell pellets are resuspended in 200 μL Lysis Buffer (0.1 Mphosphate buffer, pH 7.5, containing 0.5 mg/mL PMBS and 1.0 mg/mLLysozyme) and the lysate is used in the HTP assay as described below.

Production of Shake Flask Powders (SFP).

A shake-flask procedure was used to generate engineered pNB esterasepolypeptide powders used in secondary screening assays or in largerscale biocatalytic processes disclosed herein. Shake flask powder (SFP)includes approximately 30% total protein and accordingly provide a morepurified preparation of an engineered enzyme as compared to the celllysate used in HTP assays. A single colony of E. coli containing aplasmid encoding an engineered pNB esterase of interest is inoculatedinto 50 mL Luria Bertani broth containing 50 μg/ml chloramphenicol and1% glucose. Cells are grown overnight (at least 16 hours) in anincubator at 30° C. with shaking at 250 rpm. The culture is diluted into250 mL Terrific Broth (12 g/L bacto-tryptone, 24 g/L yeast extract, 4mL/L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO₄) containing30 μg/ml chloramphenicol, in a 1 liter flask to an optical density of600 nm (OD₆₀₀) of 0.2 and allowed to grow at 30° C. Expression of thepNB esterase gene is induced by addition ofisopropyl-β-D-thiogalactoside (“IPTG”) to a final concentration of 1 mMwhen the OD₆₀₀ of the culture is 0.6 to 0.8. Incubation is thencontinued overnight (at least 16 hours). Cells are harvested bycentrifugation (5000 rpm, 15 min, 5° C.) and the supernatant discarded.The cell pellet is resuspended with 25 mL volume of cold (5° C.) 100 mMphosphate buffer, pH 7.0, and harvested by centrifugation as above. Thewashed cells are resuspended in 12 mL of the cold phosphate buffer andpassed through a One Shot Cell Disrupter (Constant Systems Ltd.) at 40kpsi and 5° C. Cell debris is removed by centrifugation (10000 rpm, 45minutes, and 5° C.). The clear lysate supernatant is collected andstored at −20° C. Lyophilization of frozen clear lysate provides a dryshake-flask powder of crude pNB esterase polypeptide. Alternatively, thecell pellet (before or after washing) can be stored at 4° C. or −80° C.

Production of Downstream Process (DSP) Powders:

DSP powders contain approximately 80% total protein and accordinglyprovide a more purified preparation of the engineered pNB esteraseenzyme as compared to the cell lysate used in the high throughput assay.Larger-scale (˜100-120 g) fermentation of the engineered pNB esterasepolypeptides for production of DSP powders can be carried out as a shortbatch followed by a fed batch process according to standard bioprocessmethods. Briefly, pNB esterase expression is induced by addition of IPTGto a final concentration of 1 mM. Following fermentation, the cells areharvested and resuspended in 100 mM phosphate buffer, pH 7, thenmechanically disrupted by homogenization. The cell debris and nucleicacids are flocculated with polyethylenimine (PEI) and the suspensionclarified by centrifugation. The resulting clear supernatant isconcentrated using a tangential cross-flow ultrafiltration membrane toremove salts and water. The concentrated and partially purified enzymeconcentrate can then be dried in a lyophilizer and packaged (e.g., inpolyethylene containers).

Example 3: High Throughput (HTP) Screening of pNB Esterases forConversion of pNB-Protected Substrate of Compound (2) to Compound (1),Imipenem

HTP screening of cell lysates was used to guide primary selection ofengineered pNB esterase polypeptides having improved properties for theconversion of substrate compound (2) to imipenem product compound (1).

For preparing the lysates, cells were grown in 96-well plates asdescribed in Example 2 and lysates prepared by dispensing 2004 LysisBuffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mg/mL PMBS and1.0 mg/mL Lysozyme) into each well. Plates were sealed, shaken for 2 h,and then centrifuged for 20 min at 4000 rpm, 4° C., to pellet the celldebris.

HTP Assay pNB Esterase Polypeptide Activity:

A 45 μL aliquot of 0.1 M phosphate buffer at pH 7.5 and 125 μL of celllysate was added to each well of a 96-well plate. Reactions wereinitiated by adding 30 μL aliquot of a stock substrate solution (13.5g/L of compound (2) dissolved in DMF) to each well. Plates were sealed,quickly spun in the centrifuge (<1 min), and placed in shaker at 200 rpmat 15° C. for 24 h. Reactions were quenched with 800 μL of acetonitrileand samples examined by HPLC as described in Example 4.

Example 4: Analytical Procedures

HPLC Analysis of Activity of HTP Reactions

Reactions were quenched by dispensing 800 μL of acetonitrile into eachwell (as in Example 3), heat sealing the plate, shaking at high speedfor 1 min to mix, then spinning down the plate in a centrifuge at 4000rpm, 10 min, at 4° C. A 200 μL aliquot of the quenched HTP reaction wasdispensed into a 96 well round bottom plate for HPLC analysis. The 200μL samples were subject to HPLC analysis under the following conditions.

Column Poroshell EC C18, 2.6 μm, 4.6 × 100 mm with guard columnTemperature Not controlled Mobile Phase Gradient: A: Acetonitrile/0.1%formic acid; B: Water/0.1% formic acid Time (min) A % B % 0-1 2 982.8-4.0 72  28 4.2-5.0 2 98 Flow Rate 1.0 mL/min Detection 315 nm, ref400 nm Injection volume 10 μL Retention Times Compound (1), imipenem:3.1-3.16 min Compound (3) side-product (β-lactam ring opened imipenem):3.6 min Compound (2), pNB-protected imipenem: 3.8 min p-nitrobenzylalcohol: 4.1 min Side product A (ring opened diacid of compound (2)):1.2 min

Conversion of compound (2) to compound (1) was determined from theresulting chromatograms as follows:Conversion (%)=Product Area/(Product Area+Substrate Area)×100%

Example 5: Process for Conversion of Compound (2) to Compound (1) at 1mL Scale

SFP preparations of the engineered pNB esterase polypeptides were usedin 1 mL scale reactions of the conversion of a pNB-protected imipenemsubstrate of compound (2) to the product imipenem of compound (1). Thesereactions demonstrate how these biocatalysts can be used for thepreparation of carbapenem compounds such as imipenem. The reactions at 1mL scale were carried out as follows. To a 2 mL glass vial was added0.35 mL of 100 mM MES buffer (pH 7.0), 0.50 mL of a 2 g/L SFPpreparation of pNB esterase polypeptide (SEQ ID NO: 76), and 0.15 mL ofa 33.3 mg/mL solution of compound (2) in DMF. The mixture was placed ina Kuhner shaker at 200 rpm, for 2 h at 15° C. Final concentrations ofcomponents were: 5 g/L of compound (2); 15% v/v DMF; 5 g/L pNB esterasepolypeptide SFP preparation; and 100 mM MES, at pH 7.0.

Samples of 20 μL were taken at different time points and diluted with 40μL acetonitrile and shaken well. The sample was mixed with 340 μL of 100mM MES buffer, mixed well and centrifuged for 10 min. The supernatantwas analyzed by HPLC, using the instrument and parameters described inExample 4.

The HPLC time course profile results for the various pNB-esterasestested in 1 mL reactions are shown below in Table 3.

TABLE 3 % Conversion to % Conversion to pNB esterase Compound (1) @ 1 hCompound (1) @ 2 h SEQ ID NO: reaction time point reaction time point 5420 32 76 63 73 80 60 73 116  75 81

The engineered pNB esterase polypeptide of SEQ ID NO: 116 reached 75%conversion of pNB-protected compound (2) to imipenem product compound(1) at 1 h, and 80% conversion after only 2 h. The polypeptides of SEQID NO: 76 and 80 performed with slightly lower compound (2) to compound(1) conversion rates under these same conditions.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered polynucleotide encoding anengineered pNB esterase polypeptide having pNB esterase activity,wherein said pNB esterase polypeptide comprises an amino acid sequencehaving at least 90% identity to SEQ ID NO: 2 and one or more residuedifferences as compared to SEQ ID NO:2 at residue positions selectedfrom: 108, 115, 116, 130, 193, 214, 219, 273, 276, 321, and 362, whereinthe differences at positions 115 and 273 are 115Q/W and 273A/E/T/V,respectively, and optionally wherein said engineered polypeptide furthercomprises a different residue at position
 362. 2. The engineeredpolynucleotide of claim 1, wherein the amino acid residue differences ascompared to SEQ ID NO: 2 of the encoded engineered polypeptide at theresidue positions 108, 116, 130, 193, 214, 219, 276, 321, and 362 areselected from 108L/Y, 116S, 130T, 164T, 193A/D/E/V, 214G, 219A/D/L/V,276A/T/L, 321A, and 362A/D/Q/S/V.
 3. The engineered polynucleotide ofclaim 1, wherein the amino acid sequence of said encoded polypeptidecomprises one or more residue differences as compared to SEQ ID NO: 2selected from: 108L/Y, 193A/D/E/V, 219A/D/L/V, 273A/E/T/V, and362A/D/Q/S/V.
 4. The engineered polynucleotide of claim 1, wherein theamino acid sequence of said encoded polypeptide comprises a residuedifference as compared to SEQ ID NO: 2 at position 193 selected from:193A/D/E/V.
 5. The engineered polynucleotide of claim 4, wherein theamino acid sequence of said encoded polypeptide comprises a residuedifference as compared to SEQ ID NO: 2 at position 193, and wherein saidresidue is 193V.
 6. The engineered polynucleotide of claim 5, whereinthe amino acid sequence of said encoded polypeptide further comprisesresidue differences as compared to SEQ ID NO: 2 at positions 219 and 273selected from 219L/V and 273A/V.
 7. The engineered polynucleotide ofclaim 6, wherein the amino acid sequence of said encoded polypeptidefurther comprises residue differences as compared to SEQ ID NO: 2 atpositions 108 and 362 selected from 108L/Y and 362A/D/Q/S/V.
 8. Theengineered polynucleotide of claim 2, wherein the amino acid sequence ofsaid encoded polypeptide comprises a combination of residue differencesas compared to SEQ ID NO: 2 selected from: 193V, 219V, and 273A; 108Y,193D, 219V, 273A, and 362S; 108Y, 193V, 219V, 273A, and 362Q; 108Y,115Q, 193V, 219L, 273A, and 362Q; and 108Y, 115Q, 193V, 219V, 273A, and362Q.
 9. The engineered polynucleotide of claim 1, wherein the aminoacid sequence of said encoded polypeptide further comprises one or moreresidue differences as compared to SEQ ID NO: 2 selected from: 116S,130T, 164T, 214G, 276A/T/L, and 321A.
 10. The engineered polynucleotideof claim 1, wherein the amino acid sequence of said encoded polypeptidefurther comprises a residue difference as compared to SEQ ID NO: 2selected from: 49G, 94G, 96S, 227T, 251V, 267R, 271L, 274L, 313F,322C/Y, 343V, 356R, 359A, 398L, 412E, 437T, 464A, and 481R.
 11. Theengineered polynucleotide of claim 1, wherein the amino acid sequence ofsaid encoded polypeptide does not comprise a residue difference ascompared to SEQ ID NO: 2 at positions 60, 144, 317, 322, 334, 358, and370.
 12. The engineered polynucleotide of claim 1, wherein the encodedpolypeptide having pNB esterase activity has at least 1.2 fold, 2 fold,5 fold, 10 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, orgreater increased activity as compared to the polypeptide of SEQ ID NO:4 in converting compound (2) to compound (1) under suitable reactionconditions.
 13. The engineered polynucleotide of claim 1, wherein theamino acid sequence of said encoded polypeptide comprises a sequencehaving at least 90% identity to a polynucleotide sequence selected fromSEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, and
 120. 14. The engineeredpolynucleotide of claim 1 comprising a nucleotide sequence having atleast 90% sequence identity to a polynucleotide sequence selected fromSEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, and
 119. 15. An expression vectorcomprising the engineered polynucleotide of claim
 1. 16. An expressionvector comprising the engineered polynucleotide of claim
 14. 17. Theexpression vector of claim 15, further comprising at least one controlsequence.
 18. The expression vector of claim 16, further comprising atleast one control sequence.
 19. A host cell comprising the expressionvector of claim
 15. 20. A host cell comprising the expression vector ofclaim 16.